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UserGuide
OncoScan® FFPE Assay Kit
For Research Use Only.
Not for use in diagnostic procedures.
P/N 703175 Rev. 2
2
Trademarks
Affymetrix®, Axiom®, Command Console®, CytoScan®, DMET™, GeneAtlas®, GeneChip®, GeneChip–compatible™, GeneTitan®, Genotyping
Console™, myDesign™, NetAffx®, OncoScan®, Powered by Affymetrix™, PrimeView®, Procarta®, and QuantiGene® are trademarks or
registered trademarks of Affymetrix, Inc. All other trademarks are the property of their respective owners.
Limited License
Subject to the Affymetrix terms and conditions that govern your use of Affymetrix products, Affymetrix grants you a non-exclusive, nontransferable, non-sublicensable license to use this Affymetrix product only in accordance with the manual and written instructions provided
by Affymetrix. You understand and agree that except as expressly set forth in the Affymetrix terms and conditions, that no right or license to
any patent or other intellectual property owned or licensable by Affymetrix is conveyed or implied by this Affymetrix product. In particular,
no right or license is conveyed or implied to use this Affymetrix product in combination with a product not provided, licensed or specifically
recommended by Affymetrix for such use.
Patents
Cartridge Arrays: Products may be covered by one or more of the following patents: U.S. Patent Nos. 5,445,934; 5,744,305; 5,945,334;
6,140,044; 6,399,365; 6,551,817; 6,733,977; 7,629,164; 7,790,389 and D430,024 and other U.S. or foreign patents. Products are manufactured
and sold under license from OGT under 5,700,637.
Fluidics Stations: Products may be protected by one or more of the following patents: U.S. Patent Nos. 6,114,122; 6,287,850; 6,391,623;
6,422,249 and other U.S. or foreign patents.
Scanners: Products may be covered by one or more of the following patents: U.S. Patent Nos. 5,578,832; 5,631,734; 5,834,758; 5,936,324;
5,981,956; 6,025,601; 6,141,096; 6,171,793; 6,185,030; 6,201,639; 6,207,960; 6,218,803; 6,225,625; 6,252,236; 6,335,824; 6,407,858; 6,472,671;
6,490,533; 6,650,411; 6,643,015; 6,813,567; 7,682,782; 7,689,022 and other U.S. or foreign patents.
Autoloaders: Products may be covered by one or more of the following patents: U.S. Patent Nos. 6,511,277; 6,604,902; 6,705,754; 7,108,472
and other U.S. or foreign patents.
Hybridization Oven/Rotational Mixer: Products may be covered by one or more of the following patents: U.S. Patent Nos. 6,050,719;
6,386,749; 6,705,754 and other U.S. or foreign patents.
Cartridge Array Software: Products may be covered by one or more of the following patents: U.S. Patent Nos. 5,733,729; 5,795,716;
5,974,164; 6,066,454; 6,090,555; 6,185,561; 6,188,783; 6,223,127; 6,228,593; 6,229,911; 6,242,180; 6,308,170; 6,361,937; 6,420,108; 6,484,183;
6,505,125; 6510,391; 6,532,462; 6,546,340; 6,687,692; 6,607,887; 7,062,092; 7,451,047; 7,634,363; 7,674,587 and other U.S. or foreign patents.
Copyright
©2013-2014 Affymetrix Inc. All rights reserved.
Contents
Chapter 1
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
About the OncoScan® FFPE Assay Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
About This User Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Chapter 2
Laboratory Setup and Recommendations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Laboratory Setup — Two Separate Rooms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Laboratory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Pre-PCR Lab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Contamination Prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Activities in the Pre-PCR Lab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Equipment Required in Pre-PCR Lab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Post-PCR Lab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Activities in the Post-PCR Lab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Equipment Required in Post-PCR Lab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Safety Precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Chapter 3
10
10
10
11
11
12
13
13
14
14
OncoScan® Assay Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Assay and Reagent Configuration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Reagents Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
OncoScan® Assay Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Chapter 4
Best Practices for Running the OncoScan® FFPE Assay Kit Protocol . . . . . . . . 19
Laboratory Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Preparing the Work Area for Each Stage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Other Important Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Preparation & Quantification of DNA Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Handling the Normalized DNA Sample Plate
(Reaction-ready DNA Sample Plate (6.6 μL/well at 12 ng/μL)) . . . . . . . . . . . . . . . . . . . . . . .
Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Reagent Handling and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Chilling the Assay Plate Before Reagent Addition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Equipment and Calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Pipettes and Pipetting Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Seal, Vortex, and Spin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Running Gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Washing Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Thermal Cyclers, 96-Well Plate, and Adhesive Seals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Hybridization Oven . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Quality Control Gel Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Safety Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
19
19
19
20
20
20
21
22
22
22
23
24
25
25
25
26
26
27
Contents
Chapter 5
4
FFPE DNA General Requirements for OncoScan® Assay . . . . . . . . . . . . . . . . . . 28
FFPE DNA Extraction and Purification for OncoScan® Assay . . . . . . . . . . . . . . . . . . . . . . . . . 28
dsDNA Quantification Protocol for OncoScan® FFPE Samples . . . . . . . . . . . . . . . . . . . . . . . . 28
Sample Normalization (dilution to a working concentration of 12 ng/μL) . . . . . . . . . . . . . . . 29
Chapter 6
OncoScan® FFPE Assay Kit Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Preparing a Plate of Genomic DNA and a Batch Registration File . . . . . . . . . . . . . . . . . . . . . . .
Genomic DNA Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Batch Registration File Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Stage 1 — Anneal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
About this Stage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Location and Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Equipment and Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
OncoScan® Reagent Components Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Thaw Reagents and Prepare for Anneal Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Prepare the Anneal Master Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Addition of Anneal Master Mix to Anneal Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Stage 2 — Gap Fill Through 1st PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
About this Stage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Location and Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Input Required from Previous Stage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Equipment and Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
OncoScan® Reagent Components Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Thaw OncoScan® Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Prepare the AT Mix and GC Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Prepare Gap Fill Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Add Gap Fill Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Channel Split . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Add the dNTP AT Mix and dNTP GC Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Prepare and Add the Exo Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Prepare and Add the Cleavage Master Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Prepare and Add the PCR Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Stage 3 — First QC Gel and 2nd PCR (Post-PCR Lab) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
About this Stage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Location and Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Input Required from Previous Stage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Equipment and Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
OncoScan® Reagent Components Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
QC Gel Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Transfer Assay Plate to Post-PCR Lab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Preparing the 1st PCR Plate for the 2nd PCR Stage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Prepare the 2nd PCR Mix and Setup 2nd PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Prepare and Run the First QC Gel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
QC Check Point 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
30
30
30
31
31
31
31
32
32
34
35
37
37
37
37
37
38
39
39
40
41
43
46
48
49
51
54
54
54
54
54
55
55
55
56
56
59
62
Contents
Stage 4 — HaeIII Digest and Second QC Gel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
About this Stage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Location and Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Input Required from Previous Stage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Equipment and Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
OncoScan® Reagent Components Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Gel Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Thaw the Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Prepare and Run the HaeIII Digest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Prepare and Run the HaeIII Gel (Second QC Gel) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
QC Check Point 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Stage 5 — Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
About this Stage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Location and Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Input Required from Previous Stage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Equipment and Consumables Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
OncoScan® Reagent Components Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Preheat the Hybridization Ovens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Prepare the Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Prepare the Hybridization Master Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Prepare the Hyb Plate and Denature the Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Generate and Upload a Sample Batch Registration File (if it was done earlier) . . . . . . . . . . .
Load the Samples onto OncoScan® Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Chapter 7
63
63
63
63
63
64
64
64
65
68
69
70
70
70
70
70
71
71
71
72
73
76
77
Washing, Staining and Scanning Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
Equipment and Consumables Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Reagents Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Fluidics Station and Scanner Control Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Prime the Fluidics Station . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Washing and Staining Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Scanning Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Prepare the Scanner . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Prepare Arrays for Scanning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Scanning the Array . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Adding Arrays During an Autoloader Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Shutting Down the Fluidics Station . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Chapter 8
5
78
79
79
79
81
82
82
83
84
85
86
Fluidics Station Care and Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
General Fluidics Station Care . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Fluidics Station Bleach Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The Bleach Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The Rinse Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
87
87
87
90
Contents
Appendix A
6
Registering Samples in Affymetrix® GeneChip® Command Console® . . . . . . . 93
Generating a Sample Batch Registration File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Upload the Batch Registration File to AGCC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
About Batch Registration Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Using the Predefined Template — OncoScan.TEMPLATE . . . . . . . . . . . . . . . . . . . . . . . . . . .
Creating or Editing a Template . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Appendix B
Thermal Cycler Programs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
Thermal Cyclers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Pre-PCR Lab Thermal Cycler Programs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Post-PCR Lab Thermal Cycler Programs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Setting the Ramp Speed and Volume for Each Program . . . . . . . . . . . . . . . . . . . . . . . . . . .
Important Information on Using the Veriti® Thermal Cycler . . . . . . . . . . . . . . . . . . . . . . . . . .
OncoScan® Anneal Thermal Cycler Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
About the OncoScan® Anneal Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
OncoScan® Gap Fill Thermal Cycler Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
About the OncoScan® Gap Fill Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
OncoScan® 1st PCR Thermal Cycler Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
About the OncoScan® 1st PCR Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
OncoScan® 2nd PCR Thermal Cycler Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
About the OncoScan® 2nd PCR Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
OncoScan® HaeIII Digest Thermal Cycler Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
About the OncoScan® HaeIII Digest Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
OncoScan® Hybridization Thermal Cycler Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
About the OncoScan® Hybridization Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Appendix C
101
101
101
102
102
104
104
105
105
107
107
108
108
109
109
110
110
Equipment, Software, Consumables and Reagents List . . . . . . . . . . . . . . . . . 111
Required from Affymetrix® . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
From Other Suppliers — Reagents, Equipment and Consumables Required . . . . . . . . . . . . . .
Reagents Required for Gel QC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Miscellaneous Consumables Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Equipment Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Supplier Contact List . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Appendix D
93
95
97
97
97
111
113
113
114
115
116
Running QC Gels on E-Gel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
Procedure for Running First (1st PCR) and Second (HaeIII) QC Gel on E-Gel . . . . . . . . . . . . . .
Equipment, E-Gels, and Reagents Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Prepare and Run the First QC Gel (1st PCR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Prepare the Gel QC Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Prepare the Gel Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Run the E-Gel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
117
117
117
117
118
118
Appendix E
Contents
7
Prepare and Run the Second QC Gel (HaeIII Digest) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Prepare the Gel QC Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Prepare the Second QC Gel (HaeIII Digest) Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Run the E-Gel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
119
119
120
120
FFPE DNA Extraction Protocol for OncoScan® Assay . . . . . . . . . . . . . . . . . . . 122
Equipment and Reagents Required, but not Provided with OncoScan® FFPE Assay Kit . . . . . .
Equipment Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Consumables Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Reagents Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Optional Prerequisite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Preparation of Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Deparaffinization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Tissue Lysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
DNA Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
DNA Elution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Quantitation of Eluted FFPE DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Appendix F
PicoGreen® dsDNA Quantification Protocol for OncoScan® Samples . . . . . . 127
Materials Required but not Provided with OncoScan® FFPE Assay Kit . . . . . . . . . . . . . . . . . . .
Genomic DNA Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Prepare Lambda DNA Standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Prepare Sample Dilutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Prepare 1:200 Dilution of PicoGreen® Stock in 1X TE . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Perform the PicoGreen® dsDNA Quantification Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Appendix G
127
127
128
128
128
129
129
Qubit® dsDNA Quantification Protocol for OncoScan® Samples . . . . . . . . . . 130
Materials Required but not Provided with OncoScan® FFPE Assay Kit . . . . . . . . . . . . . . . . . . .
Genomic DNA Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Sample Pre-dilution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Qubit® dsDNA HS Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Normalization of Qubit®-determined FFPE DNA Concentration (Optional) . . . . . . . . . . . . .
Appendix H
122
122
123
123
123
123
124
124
125
126
126
130
130
131
131
131
132
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
General Assay Performance Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
OncoScan® FFPE Assay Kit Protocol Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
Chapter 1
Introduction
Topics in this chapter include:
 About the OncoScan® FFPE Assay Kit
 About This User Guide
About the OncoScan® FFPE Assay Kit
Obtaining genome wide copy number and loss of heterozygosity (LOH) profiles from solid tumor
samples is a significant challenge due to the difficulty of working with limited amounts of highly
modified and degraded DNA derived from heterogeneous FFPE samples.
The list of clinically relevant and actionable copy number aberrations is growing and along with this, a
recognition of the importance of genome-wide copy number and LOH profiles for solid tumor sample
analysis. Genome wide copy number can also be used to detect sub clones and clonal evolution. The
number and complexity of copy number aberrations has also been shown to be an indicator of patient
prognosis.
Traditional FFPE sample analysis techniques such as fluorescent in situ hybridization (FISH) are limited
to locus specific, low-resolution copy number information. Next generation sequencing approaches
require target preparative methods that bias copy number determination and deep coverage to provide
accurate copy number information from heterogeneous FFPE samples and may not be a practical option
for most researchers.
The OncoScan® Assay utilizes the Molecular Inversion Probe (MIP) assay technology which was
originally developed for SNP genotyping, but has subsequently been used for identifying other types of
genetic variation including focal insertions and deletions, larger copy number alterations, loss of
heterozygosity (LOH), and most recently, for somatic mutation detection.
This assay has been shown over time to perform well with highly degraded DNA, such as that derived
from FFPE- preserved tumor samples of various ages and with 100 ng DNA of starting material, thus
- making the assay a natural choice in cancer clinical research.
The OncoScan® FFPE Assay Kit is a complete and robust solution for degraded FFPE sample analysis
for solid tumor tissues. The OncoScan Assay, can be run on existing Affymetrix’ instruments, and offers
genome wide copy number coverage, with high resolution on cancer genes, plus the ability to detect
frequently tested somatic mutations with a 48 hour turnaround from DNA to results.
The optimized solution consists of:
 OncoScan® reagents and arrays
 OncoScan® Console and Nexus for Affymetrix® software
 GeneChip® Scanner 3000 7G or GeneChip® Scanner 3000 Dx2 instrumentation
Chapter 1 | Introduction
Figure 1.1 OncoScan® FFPE Solution
OncoScan™ FFPE Assay Kit
takes you from DNA to results in 48 hours!
OncoScan™ Assay & Reagents
Genomic
homology 1
Genomic
homology 2
SNP
position
MIP linear probe
Exonuclease addition
MIP probe binds to DNA
Cleavage at site #1
Nexus for Affymetrix® software
(partnership with BioDiscovery)
Amplification & digestion
at cleavage site #2
Gap fill
Biotinylated oligo
hybridized overnight
to the array
GeneChip® Scanner (GCS) 3000 7G OR
GCS 3000Dx v.2, Hyb Oven 645, FS 450 / 450 DX
About This User Guide
This manual is a guide for technical personnel conducting the Affymetrix® OncoScan® Assay
experiments in the laboratory. It contains:
 Best practices recommended by Affymetrix
 Laboratory setup
 FFPE sample preparation and quantification protocols recommended by Affymetrix
 Equipment and consumables required for each step
 Step-by-step protocols for the assay
 Protocols for washing, staining, and scanning arrays
 Fluidics Station care and maintenance
 Guidelines for processing 7, 9, 13 and 25 sample formats
9
Chapter 2
Laboratory Setup and Recommendations
This chapter provides an overview of the recommended laboratory setup to be used when performing the
Affymetrix® OncoScan® FFPE Kit Protocol.
Laboratory Setup — Two Separate Rooms
Laboratory
The use of two separate rooms greatly reduces the risk of sample contamination due to previouslyamplified PCR products. These rooms are referred to as the:
 Pre-PCR Lab
 Post-PCR Lab
For more information on laboratory requirements and the equipment required to perform this protocol,
refer to the OncoScan® FFPE Assay Kit Site Preparation Guide, P/N 703176.
The high-level steps performed in each room are presented in Table 2.1.
Table 2.1 Assay Workflow When Two Separate Rooms are Used
Room
Template
(Genomic DNA)
PCR Product
Pre-PCR Clean Lab
Assay steps:
 Genomic DNA preparation and
Picogreen quantitation of the
genomic DNA and dilution
 Anneal
 First Stage PCR
Post-PCR Lab
Assay steps:
 Second Stage PCR
 HaeIII digest
 GeneChip Array hybridization
 Array washing and staining
 Array scanning
Pre-PCR Lab
The Pre-PCR Lab should be a low copy DNA template lab, and should be free of PCR product
(amplicons). Hence, no amplified product may be taken into the Pre-PCR Lab. Do not open the seal of
the First stage PCR plate in the Pre-PCR Lab.
NOTE: The Post-PCR Lab has airborne contamination with the amplified MIP-annealed
template. It is strongly recommended not to enter the Post-PCR Lab while doing the pre-PCR
steps of the assay. (After entering the Post-Amp Lab, do not re-enter the Pre-Amp Lab without
first showering and changing into freshly laundered clothes).
Chapter 2 | Laboratory Setup and Recommendations
11
Contamination Prevention
All of the reagents and master stocks required for the steps performed in the Pre-PCR Lab should be
stored in this lab under the appropriate conditions.
2. All of the equipment required for the steps performed in this lab should be dedicated. Do not move
any equipment including ice buckets and pipettes between the Pre- and Post-PCR Labs.
3. Always wear a fresh gown, booties, hair net, and gloves to prevent PCR carryover, and to minimize
the risk of trace levels of contaminants being brought into the lab.
4. Proper gowning procedure must be followed in the Pre-PCR Lab by using the steps given below:
 Using the tacky mats to remove any dust and particulates from the bottom of shoes.
 Putting on disposable booties.
 Putting on bouffant head cover (cap).
 Putting on the right size disposable laboratory gloves.
 Putting on disposable sterile lab coats.
 Wiping down the work bench area with 70% Ethanol before and after use.
 Frequently changing the gloves throughout the assay as needed.
1.
Activities in the Pre-PCR Lab
1.
Preparation of non-amplified genomic DNA
A. Extraction and Purification of genomic DNA
B. Picogreen® quantitation of the genomic DNA
NOTE: If the Qubit® or Fluoroskan instrument is located in the Post-PCR Lab, transfer
only the volume needed for Picogreen quantitation and store the rest of the DNA in
Pre-PCR Lab.
C. Dilution or concentration of the genomic DNA as needed
2.
3.
4.
5.
6.
Anneal reaction
Gap-Fill reaction
Exonuclease digestion reaction
Cleavage reaction
First Stage PCR reaction
Chapter 2 | Laboratory Setup and Recommendations
12
Equipment Required in Pre-PCR Lab
The major pieces of equipment required for this lab are given below. Refer to Figure 2.1, Figure 2.2 and
Figure 2.3.
Figure 2.1 Pre-PCR Lab Bench Equipment Requirements
Pre-PCR Lab Equipment
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
Approved thermal cyclers, see Table 4.2 or Appendix C for validated thermal cyclers
Microfuge for 1.5 mL Eppendorf vials
Microfuge for strip tubes
Vortexer
Single/Multi- Channel Pipettes on stand [L-20, L-200, L-1000, L12-20, and L12-200]
Ice bucket
Cold blocks for 96-well PCR plates - 4 each
Plate centrifuge - refrigerated or non-refrigerated
Freezer, –20°C
Refrigerator, 2-8°C
Chapter 2 | Laboratory Setup and Recommendations
Figure 2.2 Pre-PCR Lab Bench Equipment - Recommended Setup
Figure 2.3 Pre-PCR Lab - Plate Centrifuge
Post-PCR Lab
Activities in the Post-PCR Lab
1.
2.
3.
4.
5.
6.
7.
8.
Second Stage PCR amplification
QC gel of First stage PCR by agarose gel electrophoresis
HaeIII digest of Second Stage PCR
QC gel of HaeIII digest by agarose gel electrophoresis
Preparation of hybridization target
Sample labeling and hybridization onto GeneChip® Arrays
Washing and staining of arrays
Scanning of arrays
13
Chapter 2 | Laboratory Setup and Recommendations
14
Equipment Required in Post-PCR Lab
The major pieces of equipment required for this lab are given below. The bench top equipment
requirements are same as for the Pre-PCR Lab, however additional equipment is needed for processing
QC gels. Refer to Figure 2.4.
Figure 2.4 Post-PCR Lab Bench Equipment Requirements
Post-PCR Lab Equipment
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
Approved thermal cyclers, see Table 4.2 or Appendix C for validated thermal cyclers
Microfuge for 1.5 mL Eppendorf vials
Microfuge for strip tubes
Vortexer
Single/Multi- Channel Pipettes on stand [L-20, L-200, L-1000, L12-20 (or L24-20), and L12-200
(or L24-200)
Ice bucket
Cold blocks for 96-well PCR plates - 3 each
Plate centrifuge - refrigerated or non-refrigerated
Freezer, –20°C
Refrigerator, 2-8°C
Electrophoresis gel box
Electrophoresis power supply
Gel Imager
GeneChip® Hybridization Oven 645
Fluidics Station 450 or Fluidics Station 450Dx connected to an Affymetrix® GeneChip® workstation
GeneChip® Scanner 3000 7G or GeneChip® Scanner 3000 Dx2 connected to an Affymetrix®
GeneChip® workstation
Safety Precautions
The Affymetrix® OncoScan® FFPE Assay Kit as well as the Affymetrix® OncoScan® Arrays are for
research use only. All DNA and other potentially infectious materials should be handled as if capable of
transmitting infection and disposed of with proper precautions in accordance with federal, state, and local
regulations. Some components required for this assay may pose significant health risks. Follow prudent
laboratory practices when handling and disposing of carcinogens and toxins.
Refer to the manufacturer’s Safety Data Sheet for additional information.
Chapter 3
OncoScan® Assay Overview
This chapter provides an overview of the Affymetrix® OncoScan® Assay, including information about
assay configuration and workflows.
 Assay and Reagent Configuration
 OncoScan ® Assay Workflow on page 17
Assay and Reagent Configuration
Reagents Required
For a complete list of reagents, instruments, software and consumables required, please refer to
Appendix C on page 111.
OncoScan® Reagent Kit — P/N 902294
The OncoScan® FFPE Assay Kit is designed to process 24 samples plus a negative control. (Note: the
negative control is evaluated in the gel QC steps, but is not hybridized to arrays). The OncoScan FFPE
Assay Kit may also be used to process samples in multiple runs as given in Table 3.1 and Table 3.2, below.
Table 3.1
Number of
Runs per
Assay Kit
Number of
Test
Samples per
Run
Positive
Control
Sample per
Run
Negative
Control
per Run
Total Number of
Samples
Processed per Run
Total Number of
Samples
Hybridized per
Run (excludes
negative control)
Number
of Arrays
Required
per run
Total number
of Arrays
Needed for
All Runs
1
23
1
1
25
24
48
48
2
11
1
1
13
12
24
48
3
7
1
1
9
8
16
48
4
5
1
1
7
6
12
48
Table 3.2
7 Reactions
per Run
9 Reactions
per Run
13 Reactions
per Run
25 Reactions
per Run
Number of Runs per Assay Kit
4
3
2
1
Total Number of Reagent Reactions per Assay Kit
28
27
26
25
Number of Samples (includes 1 positive control per run)
6
8
12
24
Negative Control per Run
1
1
1
1
Chapter 3 | OncoScan® Assay Overview
OncoScan Reagent Kit Components
Table 3.3 OncoScan® Reagent Kit and Components
Module
OncoScan® Somatic Mutation Probe Mix 1.0 (Pre-PCR Lab)

























*


–20°C










Wash A
Wash B
902246
902252
902251
902254
902255
902253
902256
902257
902258
902259
902260
902270







2-8°C
902249
902250
902248
902246
902269

–20°C
902247
902268

Stain 1
Stain 2
Array Holding Buffer
Individual Bottles (Post-PCR Lab)
902272

PCR Mix
Taq Polymerase (5 U/μL)
Buffer B
HaeIII Enzyme (10 U/μL)
Exo I Enzyme (20 U/μL)
Nuclease Free Water
Hybridization Mix
OncoScan® Stain Reagents (Post-PCR Lab)

–20°C
Buffer A
Gap Fill Enzyme Mix
SAP, Recombinant (1 U/μL)
dNTP Mix (A/T)
dNTP Mix (G/C)
Nuclease-free Water
Exo Mix
Cleavage Buffer
Cleavage Enzyme (2 U/μL)
PCR Mix
Taq Polymerase (5 U/μL)
OncoScan® 2nd Stage PCR and Post PCR Processing (Post-PCR Lab)
Part Number*

Positive Control (12 ng/μL)
Negative Control
Copy Number Probe Mix 1.0
Buffer A
OncoScan® Gap Fill and 1st Stage PCR (Pre-PCR Lab)

–20°C
Somatic Mutation Probe Mix 1.0
OncoScan® Copy Number Probe Mix 1.0 & Controls (Pre-PCR Lab)

Storage
902259
902260
902261
902262
902263
902253
902264
902271



902265
902266
901733
Room Temp


901680
901681
Part Numbers are for identification purposes only. Individual kit components cannot be ordered separately.
16
Chapter 3 | OncoScan® Assay Overview
17
OncoScan® Assay Workflow
This section provides an overview of the 48 hour workflow spanning 3 days, segregated by Pre-PCR and
Post-PCR Lab space.
 Day-1_ PM (Pre-PCR Lab): The DNA sample plate is prepared with the normalized DNA at
12 ng/μL. The DNA samples are then incubated overnight to anneal the MIP probe. (2-3 hours handson time depending on the number of samples, anneal overnight for 16-18 hours)
 Day-2_ AM (Pre-PCR Lab): The annealed DNA samples are processed through the Pre-PCR assay
steps; (Total time: ~2.5 to 3 hours)
 Addition of Gap-fill master mix to the overnight annealed DNA
 Divide the total volume of each reaction into two wells on different rows each containing equal
volume (10 μL), referred to as AT and GC channels (Channel Split)
 Gap-Fill the annealed probe with specific dNTP mixes
 Exonuclease reaction to remove the unligated (not gap-filled, linear) probes
 Cleavage enzyme reaction to linearize the gap-filled circular MIP Probes
 1st PCR amplification of the gap-filled, linearized MIP probe
 Day-2_ PM (Post-PCR Lab): The amplified MIP products from the 1st PCR reaction are taken
through the Post-PCR assay steps that end in Array hybridization. (Total time: ~3.5 to 4.5 hours,
depending on the number of samples and overnight Array hybridization for 16-18 hours)
 Second PCR amplification to enrich the MIP product
 HaeIII digestion of the second PCR product
 QC Gels are run to determine the size distribution of the 1st PCR reaction, and the HaeIII digested
products
 Preparation of hybridization target with the digested HaeIII product
 Denature target for hybridization
 Hybridization of target onto the OncoScan® Array
 Day-3_ AM (Post-PCR Lab): (1.5 hours to wash and 15 minutes scanning per sample (2 arrays per
sample))
 Array Wash and Stain in GeneChip Fluidics Station
 Array Scan in GeneChip Scanner
 Day-3_ PM (Post-PCR Lab): The CEL files are available for data analysis
Figure 3.1 Summary of OncoScan® Assay Workflow
Pre-PCR Lab
Anneal Samples
O/N 16-18 hrs
Annealed Samples
1st Stage:
Gap Fill
Exonuclease Cleavage
1st PCR
Amplification
3 p.m. - 5 p.m.
9 a.m. - 12 p.m.
Post-PCR Lab
1st PCR
Products
QC Gel
Stain &
Wash Arrays
2nd Stage:
2nd PCR
Scan Arrays
HaeIII
Hyb Arrays
O/N 16 - 18 hrs
1 p.m. - 5 p.m.
9 a.m. - 2 p.m.
Chapter 3 | OncoScan® Assay Overview
Figure 3.2 Detailed OncoScan® Assay Workflow
Incubation
Time
Day 1:
Day 1
Starting afternoon
+ overnight Anneal
(Hands-on time: ~ 2-3 hrs)
Prepare FFPE DNA Sample Plate
(Normalization & Sample Registration)
Pre-PCR Lab
Day 1
Anneal MIP Probe Panel to
Template DNA
Day 2 AM
Gap Fill & 1st PCR
(Pre-PCR Lab)
(Hands-on time: ~ 1.5 hrs)
Elapsed
Time
16-18 hrs
~20 hrs
Day 2:
Gap Fill Probe Ligation
22 min
Removal of Unligated Probes
30 min
Linearization of
Circularlized Probes
30 min
1st Stage PCR
35 min
23 hrs
Day 2 PM
+ Overnight Hybridization
(Hands-on time: ~ 3 hrs)
1st PCR QC
2nd Stage PCR
25min
HaeIII Digestion
1 hr & 45 min
Post-PCR Lab
Day 2
HaeIII QC Gel
Hyb Target Preparation
15 min
Hyb Target Denaturation
15 min
Begin Array Hybridization
16-18 hrs
Day 3
1.5 days
Finish mid-day Day 3
(Sample & Instrument dependent)
(Hands-on time: ~ 1.5 hrs)
Day 3:
90 min Fluidics Wash
Array Wash, Stain & Scan 7 min Scan/Array
2.5 days
18
Chapter 4
Best Practices for Running the OncoScan® FFPE Assay Kit Protocol
This section provides tips for ensuring successful performance of the protocol. Topics in this section
include:
 Laboratory Workflow on page 19
 Preparing the Work Area for Each Stage on page 19
 Other Important Guidelines on page 19
 Preparation & Quantification of DNA Samples on page 20
 Handling the Normalized DNA Sample Plate (Reaction-ready DNA Sample Plate (6.6 μL/well at 12 ng/
μL)) on page 20
 Controls on page 20
 Reagent Handling and Storage on page 21
 Chilling the Assay Plate Before Reagent Addition on page 22
 Equipment and Calibration on page 22
 Pipettes and Pipetting Recommendations on page 22
 Seal, Vortex, and Spin on page 23
 Running Gels on page 24
 Hybridization on page 25
 Washing Arrays on page 25
 Thermal Cyclers, 96-Well Plate, and Adhesive Seals on page 25
 Hybridization Oven on page 26
 Quality Control Gel Recommendations on page 26
Laboratory Workflow
The first half of the assay will take place in a Pre-PCR Lab (Anneal through 1st PCR) and the second
half will take place in the Post-PCR Lab (2nd PCR through Array Scanning).
2. Maintain a single direction workflow. It is acceptable to move from the Pre-PCR Lab to the PostPCR Lab, but moving from the Post-PCR Lab to the Pre-PCR Lab should be avoided.
3. Never bring amplified products into the Pre-PCR Lab. Never open the seal of the 1st PCR plate in
the Pre-PCR Lab. Do not spin down the 1st PCR plate in the Pre-PCR Lab.
4. Keep dedicated equipment in each room or area used for this protocol. To avoid contamination, do
not move equipment between the Pre-PCR and the Post-PCR Labs.
1.
Preparing the Work Area for Each Stage
Many of the stages in the OncoScan® FFPE Assay Kit Protocol must be performed rapidly and on ice to
carefully control enzyme activity and temperature transitions. Therefore, we recommend that you set up
all of the equipment, consumables and reagents (except for the enzymes) prior to beginning each stage.
Other Important Guidelines
During the Pre-PCR portion of the assay the thermal cycler program will be paused and resumed
multiple times. It is recommended to set an additional timer for notification so that the thermal cycler
can be paused before the program moves onto the next step.
2. It is mandatory to use compression pads on the thermal cyclers (both Veriti and ABI 9700 models)
throughout the assay in both labs.
1.
Chapter 4 | Best Practices for Running the OncoScan® FFPE Assay Kit Protocol
20
Preparation & Quantification of DNA Samples
Sample preparation and quantification method play a critical role in the success of the OncoScan® Assay.
Refer to the following sections that describe the protocols recommended by Affymetrix:
 Chapter 6, OncoScan® FFPE Assay Kit Protocol on page 30
 Appendix E, FFPE DNA Extraction Protocol for OncoScan ® Assay on page 122
 Appendix F, PicoGreen® dsDNA Quantification Protocol for OncoScan® Samples on page 127
 Appendix G, Qubit® dsDNA Quantification Protocol for OncoScan® Samples on page 130
Handling the Normalized DNA Sample Plate
(Reaction-ready DNA Sample Plate (6.6 μL/well at 12 ng/μL))
If a reaction-ready DNA sample plate was previously prepared with a volume of 6.6 μL/well at 12 ng/μL
prior to running the assay, follow the instructions below to thaw the plate.
 Thaw the reaction-ready DNA sample plate (6.6 μL/well at 12 ng/μL) at room temperature for 10 15 minutes.
 Ensure the plate seal is tight and spin the plate down at 2400 rpm for 30 seconds.
 Vortex the plate at max speed for 4 seconds and spin down at 2400 rpm for 30 seconds.
 Keep it on a cold block until ready to be used in the Anneal reaction.
Controls
Using positive and negative controls is strongly recommended to assess the performance of each run. We
recommend assaying the OncoScan® Positive Control supplied in the OncoScan® FFPE Assay Kit as a
positive control throughout the entire assay, starting from Anneal stage to Array Hybridization stage. We
recommend processing the OncoScan® Negative Control supplied in the OncoScan® FFPE Assay Kit as
a negative control for the assay through the HaeIII gel QC stage only.
Chapter 4 | Best Practices for Running the OncoScan® FFPE Assay Kit Protocol
21
Reagent Handling and Storage
Proper storage and handling of reagents is essential for robust performance. Follow these guidelines to
ensure best results:
 Use reagents that are not provided in this kit from the recommended vendors only.
 Store all reagents at the recommended temperatures and conditions. Do not use reagents that have been
improperly stored. Storage methods can profoundly impact activity.
 Upon receipt of the reagent kit, store the Affymetrix® Nuclease-free water (P/N 902253) at 4°C for
your convenience.
 Do not use expired reagents or reagents that have undergone more than the recommended number of
freeze-thaw cycles.
 Seal all vials and bottle caps well after use to prevent evaporation.
 Do not store enzymes in a frost-free freezer.
IMPORTANT: Always use the 24 reaction OncoScan® Reagent Kit for this protocol. You can
freeze/thaw the reagents in the 24 reaction kit up to 4 times.

Store the reagents used for Anneal, Gap-fill and 1st PCR only in the Pre-PCR Lab.
When Using Reagents at the Lab Bench
 Properly chill essential equipment such as cooling blocks and reagent coolers before use.
 Unless otherwise indicated, keep all reagents (except for enzymes) on ice, or in a cooling block that has
been chilled to 4°C and placed on ice during use.
 Ensure that enzymes are kept at –20°C until needed. When removed from the freezer, immediately
place in a bench top reagent cooler that has been chilled to –20°C.
 Keep all tubes, master mixes and working solutions on ice or in chilled cooling blocks on ice.
 Since enzyme activity is a function of temperature, ensure that all temperature transitions to incubation
temperatures are rapid and/or well controlled to help maintain consistency across samples.
Master Mix Preparation
Carefully follow each master mix recipe. Use pipettes that have been calibrated as per the manufacturer’s
specifications. Use only the Affymetrix Nuclease-free Water that is supplied with the kit. Do not use any
other water. If the master mix runs out during the aliquoting process on any of these procedures, it
suggests that a volume error has been made or the pipettes are not accurate. In this situation we strongly
recommend repeating the master mix preparation.
Chapter 4 | Best Practices for Running the OncoScan® FFPE Assay Kit Protocol
22
Chilling the Assay Plate Before Reagent Addition
The reagents are ALWAYS added to chilled PCR plates (placed on the cold block) throughout the assay.
Whenever a chilling step is called for, chill the plate on the cold block for 1 minute and then spin down
at 2400 rpm for 30 seconds before adding the reagents.
Equipment and Calibration
Keep dedicated equipment for this protocol in each of the lab areas, including pipettes, ice buckets,
coolers, etc. It is critical to use equipment that conforms to the guidelines and specifications detailed in
this manual. To avoid contamination, do not move equipment back and forth from the Post-PCR Lab
to the Pre-PCR Lab. Lab instrumentation plays an important role in the successful execution of this
assay. To help maintain consistency across samples and operators, all equipment must be well maintained
and routinely calibrated per manufacturer recommendations, including:
 All thermal cyclers
 GeneChip® Hybridization Oven 645
 GeneChip® Fluidics Station 450 or 450Dx
 GeneChip® Scanner 3000 7G or 3000Dx2
 Fluorescence Plate Reader or Qubit
 All single and multi-channel pipettes
Pipettes and Pipetting Recommendations
The types of pipettes specified for use throughout this protocol are:
 Single channel, manual
 12-channel, manual or electronic
 Optional: 24-channel, manual or electronic
General Pipetting Recommendations
Since the OncoScan® Assay protocol involves a series of ordered stages, the output of one stage directly
impacts the performance of the subsequent stage. Many of the reagents in the OncoScan® FFPE Assay
Kit are in very viscous solutions. For best results:
 Always use pipettes that have been calibrated as per the manufacturer’s specifications.
 It is essential that operators be proficient with the use of single and multi-channel pipettes.
 Always use filter tips for pipetting. This is essential to reduce sample contamination.
 Pipet slowly to allow enough time for the correct volume of solution to enter the pipette tip.
 Avoid excess solution on the outside of pipette tips.
 To ensure full volume transfer, check pipette tips after each pickup and dispense.
 To avoid the formation of air bubbles, dispense liquids at the bottom of each well.
 Always use the type and volume of pipette specified in the protocol.
To familiarize with the use of multi-channel pipettes, we strongly recommend practicing several times
before processing actual samples. Water can be used to get a feel for aspirating and dispensing solutions
to multiple wells simultaneously. Take special care to observe complete evacuation of liquid from all
pipette tips when using a multichannel pipette.
Chapter 4 | Best Practices for Running the OncoScan® FFPE Assay Kit Protocol
23
Electronic Pipetting Recommendations
Follow the instructions provided with the pipettes for the dispense/mix program that:
 Allows reagents to be aspirated and dispensed at a set volume.
 Mixes automatically upon dispensing, wherein the mix volume can be different from the dispense
volume.
Two options are available for tracking the number of mixes when using Rainin EDP3-Plus electronic
pipettes: the counter option, or the beep option (pipette beeps after each mix). We recommend using the
beep option, since the counter does not start at zero with each use. Instead, it counts pipette operations
sequentially. Refer to the instructions provided with your pipettes for more information.
Seal, Vortex, and Spin
Unless otherwise noted, follow the instructions below when the protocol instructs you to seal, vortex and
spin.
Handling the Plate Seal
 To minimize sample cross contamination and to ensure tight seals, use each seal only once. NEVER
REUSE A SEAL. Discard used seals immediately to avoid contaminating equipment or working
surfaces with DNA.
 The seal may become loose due to high temperature in the thermal cycler. Always ensure tight sealing
before vortexing a plate.
 Whenever a plate is taken out of the thermal cycler, before continuing on to the next step, ensure that
the seal is tight, spin the plate in the centrifuge, then remove the seal and discard.
 Whenever a plate is taken out of the freezer, first thaw the plate, ensure that the seal is tight, centrifuge,
and only then remove the plate seal.
 When reaction setup is completed, always use a new seal to seal the plate.
 When applying the seal to a plate, press the seal tightly onto the plate using an adhesive film applicator.
Using a plastic lid or a plastic tube rack is a potential source of contamination. Make sure that the seal
is tight around all plate/well edges.
Sealing Strip Tubes
Cut adhesive seal into strips wide enough to seal 8 or 12 strip tubes. Alternatively, strip caps can also be
used for sealing. Seal the strip tubes containing master mix with the adhesive strips or strip caps before
spinning in the bench top quick spin microfuge.
Vortex
Master Mix tubes: Vortex the master mix at high speed 3 times, 1 second each time.
 Vortex reagents: Vortex all reagents except the enzymes at high speed 3 times, 1 second each time.
Ensure there is no precipitate. If a precipitate is observed, repeat vortexing.
 Vortex enzyme: Quick vortex once, 1 second.
 Vortex plates: High speed for 1 second each in all corners and in the center (Figure 4.1).

Chapter 4 | Best Practices for Running the OncoScan® FFPE Assay Kit Protocol
24
Figure 4.1 Vortex Plates at the Corners and Center
NOTE: We recommend using MicroAmp® Clear Adhesive Films to seal your plates.
IMPORTANT: Always ensure that your plates are tightly sealed. A tight seal will prevent
sample loss and cross-well contamination, particularly when plates are being vortexed.
NEVER REUSE A SEAL. ALWAYS USE A NEW SEAL.
Spin
When instructed to spin down plates or reagent vials, follow these guidelines unless otherwise instructed.
 Plates:
 Spin at room temperature or at 4°C in a refrigerated centrifuge if available.
 Start the centrifuge, allow it to reach 2400 rpm and spin at that speed for 1 minute.
 Reagent Vials: 3 seconds using bench top mini-centrifuge.
 Enzyme Vials: 3 seconds using bench top mini-centrifuge.
Guidelines for All Stages of the Assay
 Pre-chill the thawed reagents on ice.
 Pre-chill empty tubes for master mix on ice and empty strip tubes in a cold block on ice.
 Leave the enzymes at –20°C until ready to use and add the enzyme as the last reagent at any step.
 All reagent additions in all steps must be performed on ice.
 Always carry the sample plate to the centrifuge or the thermal cycler on the cooling block on ice.
 Always prepare the reagents ahead for the next step. Add the reagents for the next step immediately
after chilling and spinning down the plate. Vortex, Spin down and proceed to the next step with 10
minutes of removing from the thermal cycler until the end of HaeIII step.
 Do not let the plate sit on the cold block for more than 10 minutes at any Pre-PCR assay steps
including the Cleavage Reaction step.
 Do not let the plate sit on the thermal cycler for more than 30 minutes at the PCR and HaeIII steps.
Stopping Points on the Assay
 First PCR plate can be frozen if the assay can not be continued.
 HaeIII plate can be frozen at the end of HaeIII reaction, if the assay can not be continued. (DO NOT
ADD hybridization cocktail to the HaeIII plate.)
Running Gels

3% Pre-cast Agarose 96-well format gels: Run gels at 150V for 15 minutes in the appropriate
electrophoresis chamber.
Chapter 4 | Best Practices for Running the OncoScan® FFPE Assay Kit Protocol
25
Hybridization


Load only 6 to 8 arrays at a time. Remove the seal from the hybridization plate for only 6-8 samples at
a time.
Preheat the hybridization oven to 49°C at least one hour prior to use.
Washing Arrays
It is important to work quickly when processing arrays for washing. Delays during this step will impact
data quality. To optimize this step, we suggest the following:
 30 minutes before hybridization is complete, prime the fluidics stations with the correct wash buffers.
Start the Fluidics Protocol and follow the directions on the LCD panel of the fluidics station.
 Load Stain 1, Stain 2, and the Array Holding buffer in their respective positions on the fluidics station.
Eject the wash block to avoid sensor time out.
 Process only 6-8 arrays at a time.
 Minimize delays when performing all steps after the arrays are removed from the oven, up to the time
when washing begins.
Thermal Cyclers, 96-Well Plate, and Adhesive Seals
To run the OncoScan FFPE™ Assay Kit Protocol at a throughput of 48 assays/day, you will need 2
thermal cyclers: 1 in the Pre-PCR Lab; 1 in the Post-PCR Lab.
The OncoScan FFPE Assay Kit Protocol has been optimized using the following thermal cyclers, 96well plate, and adhesive films.
IMPORTANT: Use only the 96-well plate and adhesive seals listed in Table 4.1, and only the
thermal cyclers listed in Table 4.2. Using other plates and seals that are incompatible with
these thermal cyclers can result in loss of sample or poor results.
Please refer to Appendix B for critical information about programing and operating Veriti
Thermal Cyclers.
Table 4.1 96-Well Plate and Adhesive Seals
Item
96-well Half-skirted PCR Plate
MicroAmp® Clear Adhesive Film
Vendor
Part Number
E&K Scientific
289196
Applied Biosystems
4306311
Table 4.2 Thermal Cyclers
Laboratory
Thermal Cyclers Validated for Use
Pre-PCR Clean Area
Use one of these units.
Applied Biosystems Units:
®
 GeneAmp PCR System 9700 (silver block or gold-plated
silver block), P/N 4314878
®
 Veriti 96-Well Thermal Cycler, 0.2 mL, P/N 4375786
Post-PCR Area
Use one of these units
Applied Biosystems Units:
®
 GeneAmp PCR System 9700 (silver block or gold-plated
silver block), P/N 4314878
®
 Veriti 96-Well Thermal Cycler, 0.2 mL, P/N 4375786
Chapter 4 | Best Practices for Running the OncoScan® FFPE Assay Kit Protocol
26
Program Your Thermal Cyclers
Use only calibrated thermal cyclers. We recommend that thermal cyclers be serviced at least once per year
to ensure that they are operating within the manufacturer’s specifications. The thermal cycler programs
listed below are used in this protocol. Enter and save the following programs prior to processing samples
on the appropriate thermal cycler in the Pre-PCR Lab and Post-PCR Lab.
Thermal cycler program details are listed in Appendix B, Thermal Cycler Programs on page 101. For Veriti
Thermal Cyclers, please pay attention to the information in the section, Important Information on Using
the Veriti® Thermal Cycler on page 102 regarding programming and operating the Veriti Thermal Cycler
BEFORE creating any programs.
Pre-PCR Lab Thermal Cycler Programs
Set up the thermal cyclers in the Pre-PCR Lab to run the following programs:
 OncoScan Anneal
 OncoScan Gap Fill
 OncoScan 1st PCR
Post-PCR Lab Thermal Cycler Programs
Set up the thermal cyclers in the Post-PCR Lab to run the following programs:
 OncoScan 2nd PCR
 OncoScan HaeIII
 OncoScan Hybridization
Hybridization Oven
Confirm that the GeneChip® Hybridization Oven 645 is calibrated before starting the hybridization
step. Accurate hybridization temperature is critical for this assay. We recommend servicing hybridization
ovens at least once per year to ensure that they are operating within the manufacturer's specifications.
Quality Control Gel Recommendations
We recommend running two quality control gels during the execution of this assay protocol. Knowing in
advance that a sample is unlikely to provide data of acceptable quality through in-process QC failures
will save arrays. The purpose of each gel is described below.
 Gel 1, 1st Stage PCR: Run to identify any samples that did not amplify. For samples in which
successful amplification has occurred, one single band at approximately120 base pairs should be seen.
No distinct band at approximately 120 base pairs should be visible in the Negative Control and in
samples in which amplification has not occurred. A smear or another unexpected band indicates the
PCR did not work. These samples should not be processed for Array Hybridization.
 Gel 2, HaeIII Digest: Run after HaeIII digestion at 37°C to confirm acceptable gel banding pattern.
The gel indicates both successful amplification during the second stage PCR reaction and HaeIII
digestion. The predominant pattern should be two bands at approximately 40 and 70 bp.
NOTE: If the QC gel was run at the end of the HaeIII digest thermal cycler program (after the
deactivation of the enzyme at 95°C), a smear, around 50-100 bp, will be observed instead of
the two bands (40 bp and 70 bp). Hence, it is strongly recommended to run the HaeIII digest
gel at the end of 37°C.
Chapter 4 | Best Practices for Running the OncoScan® FFPE Assay Kit Protocol
27
Safety Information
WARNING: For research use only. Not recommended or intended for diagnosis of disease in
humans or animals. Do not use internally or externally in humans or animals.
CAUTION: All chemicals should be considered as potentially hazardous. We therefore
recommend that this product is handled only by those persons who have been trained in
laboratory techniques and that it is used in accordance with the principles of good laboratory
practice. Wear suitable protective clothing, such as lab coat, safety glasses and gloves. Care
should be taken to avoid contact with skin and eyes. In case of contact with skin or eyes, wash
immediately with water. See SDS (Safety Data Sheet) for specific advice.
Chapter 5
FFPE DNA General Requirements for OncoScan® Assay
The general requirements for the FFPE DNA extraction, purification and quantification methods are
described in this chapter. The success of this assay requires accurate quantification of the FFPE DNA by
PicoGreen® quantification method. Hence, Affymetrix strongly recommends using the tested protocols
provided below. Affymetrix also recommends using the OncoScan® Positive Control supplied in the
OncoScan® Reagent Kit (Module P/N: 902268, Reagent P/N: 902249) as a positive control carried
through the entire assay from Anneal up to hybridization on the arrays.
FFPE DNA Extraction and Purification for OncoScan® Assay
IMPORTANT: Affymetrix strongly recommends using the QIAamp® DNA FFPE Tissue Kit
protocol for purifying DNA from FFPE Blocks that will be used in the OncoScan® Assay. For
improved DNA yields, we also recommend a modification to the QIAamp DNA FFPE Tissue Kit
protocol. The modified procedure adds a heating step at 98°C for 15 minutes to improve the
tissue digestion process to release DNA from tissue sections.
The protocol found in Appendix E, FFPE DNA Extraction Protocol for OncoScan® Assay is required and
critical to generate good quality samples for the OncoScan® Assay. DNA must be eluted using the elution
reagent provided in the recommended kit (ATE buffer). DNA MUST NOT BE eluted in Water.
Other optional tested elution reagents:
 1x TE Buffer with low EDTA at pH 8.0 (with 10 mM Tris at pH 8.0 and 0.1 mM EDTA)
Please refer to the QIAamp DNA FFPE Tissue Kit protocol for more information on the QIAGEN web
page given below.
http://www.qiagen.com/Products/Catalog/Sample-Technologies/DNA-Sample-Technologies/
Genomic-DNA/QIAamp-DNA-FFPE-Tissue-Kit#resources
NOTE: Affymetrix highly discourages the use of any sample preparation that is not silica
based in OncoScan® FFPE Assay.
dsDNA Quantification Protocol for OncoScan® FFPE Samples
IMPORTANT: It is mandatory to determine the sample concentration using a dsDNA specific
quantification method. We strongly recommend using either Quanit-iT™ PicoGreen Assay or
Qubit dsDNA Quantification, both by Life Technologies, as both of these methods have been
validated for use in the OncoScan® FFPE Assay. Sample concentration determined by UV
absorbance or NanoDrop must not be used at all in this assay.
Perform either of the two Affymetrix recommended and tested dsDNA quantitation protocols to measure
the concentration of the eluted FFPE DNA. These protocols are provided in:
 Appendix F, PicoGreen® dsDNA Quantification Protocol for OncoScan® Samples on page 127
 Appendix G, Qubit® dsDNA Quantification Protocol for OncoScan® Samples on page 130,
NOTE: There are other dsDNA quantitation kits available that may deliver results different
than the above mentioned kits. Hence, Affymetrix strongly recommends not using any kit
other than the ones recommended above.
Chapter 5 | FFPE DNA General Requirements for OncoScan® Assay
29
Sample Normalization (dilution to a working concentration of 12 ng/μL)
All genomic DNA samples should be normalized to a single concentration of 12 ng/μL using low EDTA
1X TE buffer (10mM Tris, 0.1mM EDTA, pH8.0). The positive control DNA that is included in the
OncoScan® FFPE Assay Kit is already normalized to a working concentration of ~12 ng/μL.
Preparing a Plate of Normalized FFPE Genomic DNA
A normalization plate is generated so that all DNA samples used in the assay are at the required volume
(6.6 μL) and concentration (12 ng/μL) from the Stock DNA sample plate or Stock DNA vials.
Handling the Stock (original concentration) DNA Sample Plate for Normalization:
The steps provided below describe how to process a stock (original) DNA plate for sample normalization
if the samples were already distributed in a “stock DNA sample plate”. This is to ensure a complete
resuspension of the DNA samples.
1.
2.
3.
4.
5.
6.
7.
8.
Thaw the stock DNA sample plate at room temperature for 15 minutes.
Ensure the plate seal is tight and spin the plate down at 2400 rpm for 30 seconds.
Vortex the plate at max speed for 4 seconds and spin down at 2400 rpm for 30 seconds.
Repeat Step 2 and Step 3 above.
Keep it on the cold block until ready to be used for preparing the normalized DNA plate.
When preparing the dilution or normalization plate, add the volume of reduced EDTA TE buffer
(10mM Tris, pH8.0, 0.1mM EDTA, pH8.0) first before adding the volume of the stock DNA.
After addition of both reduced EDTA TE buffer and DNA, tightly seal and vortex the plate at max
speed for 4 seconds. Spin the plate down at 2400 rpm for 30 seconds.
Keep the plate on the cold block until ready to be used in the Anneal reaction
Handling the Stock (original concentration) DNA Sample Vials for Normalization:
The steps provided below describe how to process the stock DNA vials for sample normalization if the
samples were stored in vials at –20°C. This is to ensure a complete resuspension of the DNA samples.
1.
2.
3.
4.
5.
6.
7.
Thaw the DNA vials at room temperature for 15 minutes.
Vortex the vials at max speed for 4 seconds and spin down briefly.
Leave the vials at room temperature for 5 minutes and then repeat Step 2, above, again.
Keep the vials on ice until ready to be used for sample normalization.
When preparing the dilution or normalization plate, add the volume of low EDTA TE buffer
(10mM Tris, pH8.0, 0.1mM EDTA, pH8.0) first before adding the volume of the stock DNA.
After addition of both reduced EDTA TE buffer and DNA, tightly seal and vortex the normalized
sample plate (or vials) at max speed for 4 seconds.
Spin down the plate briefly and keep on ice until ready to be used in the Anneal reaction.
Chapter 6
OncoScan® FFPE Assay Kit Protocol
The OncoScan® FFPE Assay Kit Protocol is presented in stages. The stages are:
 Stage 1 — Anneal
 Stage 2 — Gap Fill Through 1st PCR
 Stage 3 — First QC Gel and 2nd PCR (Post-PCR Lab)
 Stage 4 — HaeIII Digest and Second QC Gel
 Stage 5 — Hybridization
 Chapter 7, Washing, Staining and Scanning Arrays
Preparing a Plate of Genomic DNA and a Batch Registration File
Genomic DNA Preparation
To perform the OncoScan® FFPE Assay Kit Protocol, you will need to prepare a plate of genomic DNA
(gDNA). The protocol is written for 24 samples (23 samples and 1 positive control) plus 1 negative
control.
As Part of gDNA Plate Preparation:
Determine sample concentrations (we recommend using the Quant-iT™ PicoGreen® dsDNA Assay Kit
from Life Technologies). Refer to dsDNA Quantification Protocol for OncoScan® FFPE Samples on
page 28.
2. Normalize all gDNA samples to a single concentration of 12 ng/μL using 1X TE (pH 8.0) with
Reduced EDTA (0.1 mM EDTA). Refer to Sample Normalization (dilution to a working concentration
of 12 ng/μL) on page 29.
The OncoScan Positive Control included in the OncoScan® Reagent Kit has already been normalized
to a working concentration based on the concentration determined by PicoGreen quantification.
1.
NOTE: Positive Control DNA may be retired from time to time as dictated by availability. New
Positive Control DNA will be introduced if this occurs.
IMPORTANT: We strongly recommend you determine your sample concentration using the
Quant-IT PicoGreen Assay by Life Technologies. Sample concentration determined by UV
absorbance should not be used at all in this assay.
Batch Registration File Preparation
Sample information can be recorded in a Batch Registration File and uploaded into Affymetrix®
GeneChip® Command Console® (AGCC). It is recommended to enter the sample information into a
batch registration file when preparing the genomic DNA plate. During array preparation, array barcodes
must be added to this file prior to uploading the information into AGCC.
Instructions on completing the sample information spreadsheet are listed in Appendix A, Registering
Samples in Affymetrix® GeneChip® Command Console® on page 93.
Chapter 6 | OncoScan® FFPE Assay Kit Protocol
31
Stage 1 — Anneal
About this Stage
This stage begins in the Pre-PCR Lab. During this stage, genomic DNA samples and controls, and
reagents (Anneal Master Mix) are combined in an Anneal Plate.
The Anneal Plate is then placed on a thermal cycler and the program, OncoScan Anneal, is run. Because
the samples must be left to anneal for 16 to 18 hr, this stage is typically performed at the end of the day,
and the program is allowed to run overnight.
Location and Duration



Pre-PCR Lab
Hands-on time: approximately 45 min
Thermal cycler time: 16 to 18 hr
Equipment and Materials Required
The following equipment and materials are required to perform this stage.
In the Pre-PCR Lab
Table 6.1 Equipment and Materials Required in Pre-PCR Lab for Stage 1 — Anneal
Quantity
Item
2
Cold block, 96-well, chilled in 4°C refrigerator
1
Centrifuge, plate
1
Eppendorf Nuclease-free tubes, 1.5 mL
As required
Strip tubes, strip caps
1
Ice container, rectangular, filled with ice
1
Marking pen, extra fine point, permanent
As required
MicroAmp Clear Adhesive Films
1
Microcentrifuge for vials
1
Microfuge for strip tubes
1
Half-skirt 96-well plate
1 each
As required
Pipette:
 Single-channel P20
 Single-channel P200
 Single-channel P1000
 12-channel P200
 12- or 24-channel P20
Pipette tips for the pipettes listed above
1
Thermal Cycler, 96-well GeneAmp PCR System 9700 (gold or silver block) or Veriti Thermal Cycler
1
Compression Pad for Thermal Cycler
1
Vortexer
5-23
Customer dependent DNA
Chapter 6 | OncoScan® FFPE Assay Kit Protocol
32
OncoScan® Reagent Components Required
Table 6.2 OncoScan® Reagent Components Required
OncoScan® Reagents from Module OncoScan Somatic Mutation Probe Mix 1.0
Somatic Mutation Probe Mix 1.0
Table 6.3 OncoScan® Reagent Components Required
OncoScan® Reagents from Module OncoScan Copy Number Probe Mix 1.0 & Controls
Copy Number Probe Mix 1.0
Positive Control
Negative Control
Buffer A
Thaw Reagents and Prepare for Anneal Program
Location: Pre-PCR Lab
Ensure the thermal cycler is setup to run the program at the appropriate ramp speed (max for the 9700
and 9700-Max-Mode setting for the Veriti Thermal Cycler. Refer to Ramp Speed Settings for Veriti®
Thermal Cycler on page 102.
2. Ensure all 3 programs (OncoScan Anneal, OncoScan Gapfill, OncoScan 1stPCR) have been created in the
thermal cycler.
1.
NOTE: Refer to Appendix B, Setting the Ramp Speed and Volume for Each Program for
detailed instructions. For Veriti Thermal Cyclers, refer to the information on converting a
method to create a method for the Veriti Thermal Cycler with ramp rates that simulate
those on the 9700 Thermal Cycler at Max mode.
3.
If the DNA sample plate has been prepared as an Anneal-ready plate (containing 6.6 μL of DNA per
well at 12 ng/μL), do not transfer the samples to a new Anneal plate which could result in sample
loss. Label the same sample plate as “Anneal” and mark the sample wells (Row A, or Rows A & E)
as specified in the OncoScan® FFPE Assay Kit Quick Reference Card, (7 Sample QRC, P/N 703177;
9 Sample QRC, P/N 703193; 13 Sample QRC, P/N 703188; 25 Sample QRC, P/N 703189).
Chapter 6 | OncoScan® FFPE Assay Kit Protocol
4.
33
The Master Mix preparation tables throughout the assay contain the information on the appropriate
volume of the reagent for processing 7, 9, 13 or 25 reactions in columns 4, 5, 6, and 7 respectively.
Hence,
 For processing 7 reactions (5 gDNA samples plus 1 positive control and 1 negative control), use
information from column-4 in the reagent master mix tables at each step of the assay. This column
will provide the reagent volume needed for processing 7 anneal reactions, 14 Post-GapFill reactions
after channel-split and hybridizing 12 arrays.
 For processing 9 reactions (7 gDNA samples plus 1 positive control and 1 negative control), use
information from column-5 in the reagent master mix tables at each step of the assay. This column
will provide the reagent volume needed for processing 9 anneal reactions, 18 Post-GapFill reactions
after channel-split and hybridizing 16 arrays.
 For processing 13 reactions (11 gDNA samples plus 1 positive control and 1 negative control), use
information from column-6 in the reagent master mix tables at each step of the assay. This column
will provide the reagent volume needed for processing 13 anneal reactions, 26 Post-GapFill
reactions after channel-split and hybridizing 24 arrays.
 For processing 25 reactions (23 gDNA samples plus 1 positive control and 1 negative control), use
information from column-7 in the reagent master mix tables at each step of the assay. This column
will provide the reagent volume needed for processing 25 anneal reactions, 50 Post-GapFill
reactions after channel-split and hybridizing 48 arrays.
Table 6.4
Number of DNA
Samples Processed
Number of Tubes per Tube Strip
Needed to Aliquot Master Mix
Master Mix Column
(from the table) to be Used
7 Samples
7 Tube Strip
Column 4
9 Samples
9 Tube Strip
Column 5
13 Samples
12 Tube Strip
Column 6
25 Samples
12 Tube Strip
Column 7
Prepare for Anneal Program
Turn on a thermal cycler and allow the lid to heat up.
Setup an ice pan and place two cold blocks on ice.
3. Label a 96-well PCR plate ANN that will be used for Anneal. Place it on a cold block and keep it
covered.
1.
2.
Thawing the DNA Sample Plate (Normalized at 12 ng/μL)
Remove the DNA sample plate (prepared in section Genomic DNA Preparation on page 30 and has
been normalized to 12 ng/μL) from –20°C and thaw at room temperature for 15-30 minutes. Ensure
the seal is tight.
2. Once the DNA has been completely thawed, vortex the plate at max speed for 5 seconds and spin the
sample plate and place it on the cold block.
3. Aliquot 6.6 μL of the DNA to the “ANNEAL” plate in the corresponding wells of the Anneal Plate
as entered in the Batch Registration File.
4. Mark the reaction wells with a permanent marker for easier addition of reagents. Keep the plate
covered with a plate sealing film on a cold block until ready to use.
1.
Chapter 6 | OncoScan® FFPE Assay Kit Protocol
34
To thaw the reagents:
Remove the following reagents from the OncoScan® Reagent Kit.
 Buffer A
 Copy Number Probe Mix 1.0
 Somatic Mutation Probe Mix 1.0
2. Thaw them at room temperature for 10-15 minutes.
3. After the vials have been completely thawed, vortex them at max speed for 3 seconds, spin down
briefly and place them on ice.
1.
Prepare the Anneal Master Mix
Location: Pre-PCR Lab
To Prepare the Anneal Master Mix:
1.
2.
3.
4.
5.
6.
Label a 1.5 mL Eppendorf tube with Ann.
Place a tube strip as required by the number of samples in a cold block and the Ann Eppendorf tube
on ice until ready to use.
To the tube labeled Ann, add the reagents listed in Table 6.5 in the order shown.
Vortex between additions. Vortex entire Anneal Master Mix for 3 sec at max speed and spin down
briefly.
Using the column appropriate to the number of reactions, aliquot the specific volumes of Anneal
Master Mix from Table 6.5 to a corresponding tube strip.
Keep the aliquoted strip tube covered on a cold block until ready to use.
Table 6.5 Anneal Master Mix
Number of Runs per Assay Kit
4
3
2
1
Number of Samples (includes 1 positive control per run)
6
8
12
24
Negative Control per Run
1
1
1
1
Reagent
P/N
1 Reaction
7 Reactions
9 Reaction
13 Reaction
25 Reaction
(~60% overage)
(~60% overage)s
(~60% overage)
(~60% overage)
Buffer A
902246
1.53 μL
17.1 μL
22.0 μL
31.8 μL
61.2 μL
Copy Number Probe Mix 1.0
902248
1.37 μL
15.3 μL
19.7 μL
28.5 μL
54.8 μL
Somatic Mutation Mix 1.0
902247
0.5 μL
5.6 μL
7.2 μL
10.4 μL
20.0 μL
3.40 μL
38.1 μL
49.0 μL
70.7 μL
136.0 μL
5.0 μL
7-Tube Strip
5.0 μL
9-Tube Strip
5.0 μL
12-Tube Strip
10 μL
12-Tube Strip
Total Volume
Volume per Tube for Tube Strip
Chapter 6 | OncoScan® FFPE Assay Kit Protocol
35
Addition of Anneal Master Mix to Anneal Plate
Location: Pre-PCR Lab
To Transfer Anneal Master Mix to Samples in Anneal Plate:
1.
Using a 12-channel P20 pipette, transfer 3.4 μL of Anneal Master Mix from the strip tube to specific
rows depending on the sample size being processed on the Anneal Plate. Pipet up and down 3 times
to rinse tips (Figure 6.1).
NOTE: For the 13 or 25 Sample Reaction, add 3.4 μL of Anneal Master Mix from the Master
Mix tube to the Negative Control sample (Figure 6.1).
NOTE: Now the current volume in the Anneal Plate is 10 μL.
Figure 6.1 Adding Anneal Master Mix to Anneal Plate
7 Samples - 10 μL
A
1
2
3
4
5
+ _
6
7
8
13 Samples - 10 μL
9
10
11
ANN
12
A
B
B
C
C
D
D
E
E
F
F
G
G
H
H
1
2
9 Samples - 10 μL
A
1
2
3
4
5
6
7
+ _
8
4
5
6
7
8
9
10
11
ANN
12
+
25 Samples - 10 μL
9
10
11
ANN
12
B
C
C
D
D
E
E
F
F
G
G
H
H
= Negative Control
1
A
B
= Positive Control
3
_
= Sample
2
3
4
5
6
7
8
9
10
11
+
_
= Empty well
ANN
12
= Master Mix tube
Tightly seal the Anneal Plate with a clear adhesive film. Vortex the Anneal Plate at max speed for
5 seconds using the 5-point plate vortexing method. Spin down the plate at 2400 rpm for 60 seconds.
3. Place the plate on the thermal cycler and start the OncoScan Anneal program.
4. After 6 minutes into the run, pause the program at 58°C (1 min into 58°C). Remove the ANNEAL
plate from the thermal cycler and place on a chilled cold block on ice for 1 minute. Ensure the seal is
tight.
2.
Chapter 6 | OncoScan® FFPE Assay Kit Protocol
36
Spin down the plate at 2400 rpm for 30 seconds.
6. Place the ANNEAL plate back on the thermal cycler and press Start one more time to Resume.
5.
NOTE: It is good practice to double check that the thermal cycler program did resume.
Check all programs after pressing Resume to make sure the program is indeed running.
7.
Incubate the samples for 16 to 18 hours. Do not incubate samples for more than 18 hours.
Figure 6.2 OncoScan Anneal Thermal Cycler Program
95°C
5 min
58°C
16 - 18 hr
58°C
∞
Pause the cycler,
chill for 1 min and
Spin plates
IMPORTANT: Throughout the protocol, when carrying any 96 well plate to and from the
centrifuge, carry the plate in the chilled cold block to keep the reactions as cold as possible.
Chapter 6 | OncoScan® FFPE Assay Kit Protocol
37
Stage 2 — Gap Fill Through 1st PCR
About this Stage
During this stage, Gap Fill Mix is added to each reaction. Then the samples are transferred from the
Anneal Plate to 1st PCR Plate.
The 1st PCR Plate is then placed on a thermal cycler and the program OncoScan Gap Fill is started.
During the first 82 min of this program, five additional reagents are added to the Assay Plate, one reagent
at a time. Prior to each addition, the plate is removed from the thermal cycler and cooled on the cold
block for 1 min and spun down at 2400 rpm for 30 sec.
The OncoScan® Reagents that the operator is adding during thermal cycling are found in the module
Gap Fill and 1st stage PCR:
1.
2.
3.
4.
5.
Gap Fill Mix
dNTP Mix
Exo Mix
Cleavage Mix
PCR Mix
Location and Duration



Pre-PCR Lab
Hands-on time: 1 hour 15 min
Thermal cycler time: 1 hr 20 min
Input Required from Previous Stage
Table 6.6
Item
Anneal Plate
Equipment and Materials Required
Table 6.7 Equipment and Materials Required for Stage 2 — Gap Fill Through 1st PCR
Quantity
Item
4
Cold block, chilled in 4°C refrigerator
1
Anneal Plate from previous stage
1
Centrifuge, plate
1
Ice container, rectangular, filled with ice
As required
MicroAmp Clear Adhesive Films
As required
Microcentrifuge for tubes and strips
Chapter 6 | OncoScan® FFPE Assay Kit Protocol
Table 6.7 Equipment and Materials Required for Stage 2 — Gap Fill Through 1st PCR (Continued)
Quantity
1 each
Item
Pipettes:
Single-channel P20
 Single-channel P200
 Single-channel P1000
 12-channel P200
 12- or 24-channel P20

As required
Filtered Pipette tips fitted for the above pipettes
1
Half-skirt PCR Plate
1
Bench top Cooler
1
Thermal Cyclers, 96-well GeneAmp PCR System 9700 (gold or silver block),
Veriti Thermal Cycler
As required
Tubes, Eppendorf 2.0 mL, Nuclease-free
As required
Tubes, Eppendorf 1.5 mL, Nuclease-free
4
Tube strips with caps, PCR 12-well
1
Blue Permanent Marker, Extra fine tip
1
Red Permanent Marker, Extra fine tip
1
Timer
OncoScan® Reagent Components Required
Table 6.8 OncoScan® Reagent Components Required
OncoScan Reagents from Module OncoScan Gap Fill and 1st Stage PCR
Nuclease-free Water
Buffer A
Gap Fill Enzyme Mix
SAP, Recombinant
dNTP Mix (AT), dNTP Mix (GC)
Exo Mix
Cleavage Buffer
Cleavage Enzyme
PCR Mix
Taq Polymerase
38
Chapter 6 | OncoScan® FFPE Assay Kit Protocol
39
Thaw OncoScan® Reagents
IMPORTANT: Leave the Exo Mix, Cleavage Enzyme, Gap Fill, SAP and the Taq Polymerase at
–20°C until ready to use.
To Thaw the Reagents:
Place the Buffer A, dNTP AT Mix and dNTP GC Mix, Cleavage Buffer, water and PCR Mix on
the bench top at room temperature for 10-15 minutes to thaw.
2. Vortex all of the reagents at max speed for 3-6 seconds, spin down and keep on ice until ready to use.
1.
NOTE: It is highly recommended to prepare the AT Mix and GC Mix separately and using a
new pair of gloves for each mix. It is also useful to label all wells, tubes and strips for dNTP
AT mix in a different colored lab marker relative to the dNTP GC mix. This will help in easy
identification of the different dNTP mixes and avoid a mix-up.
Prepare the AT Mix and GC Mix
To Prepare the AT Mix and GC Mix
Place 4 chilled cold blocks in a rectangular ice bucket filled to the rim with ice.
Figure 6.3 Cold Blocks in Ice Bucket
NOTE: Avoid any type of contamination between the AT and GC mixes during preparation.
Mark the tubes and strips as specified below and keep them in separate cold blocks.
1.
2.
3.
4.
5.
Label two 1.5 mL Eppendorf tubes for each dNTP master mix as follows: label one tube AT with a
blue colored marker and the other tube GC with a red colored marker. Place these tubes on ice until
ready to use.
Label two sets of strip tubes as follows: label one set of strip tubes AT in blue colored marker and the
other set of strip tubes GC in red colored marker. Place both sets of labeled strip tubes in a cold block
on ice until ready to use.
To the tube labeled AT, use the appropriate column in Table 6.9 to add the reagents in the order
shown. Rinse the pipette tips three times.
Vortex the entire AT Master Mix tube for 3 sec at maximum speed. Spin down briefly in microfuge.
Using the column appropriate to the number of reactions from Table 6.9, aliquot the specific volumes
of the AT Mix from Table 6.9 to the strip tubes labeled AT. Cover the strip tube with caps or a strip
from the MicroAmp adhesive film. Spin down the strip tubes if needed in case of bubbles.
Chapter 6 | OncoScan® FFPE Assay Kit Protocol
6.
40
Keep the strip tube on chilled cold blocks until ready to use.
NOTE: In Table 6.9 and Table 6.10, you will notice the master mix volumes are the same
for the 7, 9 and 13 sample workflows. Irrespective of the number of samples processed,
the A/T and G/C master mixes are always prepared for 24 samples, due to the small
volume of the reagents used in master mix preparation. Discard any leftover master mixes
and always prepare them fresh on the day of Gap Fill.
Table 6.9 A/T Mix
Reagent
P/N
1 Reaction
7 Reactions
9 Reactions
13 Reactions
25 Reactions
(~20% overage)
(~20% overage)
(~20% overage)
(~20% overage)
Water
902253
3.93 μL
113 μL
113 μL
113 μL
118 μL
dNTPs (A/T)
902254
0.07 μL
2.1 μL
2.1 μL
2.1 μL
2.2 μL
4.0 μL
115 μL
115 μL
115 μL
120 μL
9.6 μL
7-Tube Strip
9.6 μL
9-Tube Strip
9.6 μL
12-Tube Strip
9.6 μL
12-Tube Strip
Total
Volume per Tube for Tube Strip
NOTE: CHANGE GLOVES.
contamination.
Use fresh gloves while preparing
the G/C
mix to avoid
To the tube labeled GC, using the column appropriate to the number of reactions add the reagents
listed in Table 6.10 in the order shown. Rinse the pipette tips three times.
8. Vortex the entire GC Master Mix tube for 3 sec each at maximum speed. Spin down briefly at
2400 rpm.
9. Using the column appropriate to the number of reactions from Table 6.10, aliquot the specific
volumes of the GC Mix from Table 6.10 to the strip tube labeled GC. Cover the strip tube with caps
or strips of plate seal. Spin down the strip tube if needed in case of bubbles.
10. Keep the strip tube on chilled cold blocks until ready to use.
7.
Table 6.10 G/C Mix
Reagent
P/N
1 Reaction
7 Reactions
9 Reactions
13 Reactions
25 Reactions
(~20% overage)
(~20% overage)
(~20% overage)
(~20% overage)
Water
902253
3.93 μL
113 μL
113 μL
113 μL
118 μL
dNTPs (G/C)
902255
0.07 μL
2.1 μL
2.1 μL
2.1 μL
2.2 μL
4.0 μL
115 μL
115 μL
115 μL
120 μL
9.6 μL
7-Tube Strip
9.6 μL
9-Tube Strip
9.6 μL
12-Tube Strip
9.6 μL
12-Tube Strip
Total
Volume per Tube for Tube Strip
Prepare Gap Fill Mix
To Prepare the Gap Fill Mix:
NOTE: CHANGE GLOVES. Use fresh gloves to prepare the Gap-Fill Master Mix.
Label a 1.5 mL Eppendorf tube and one set of strip tubes with the letter G.
2. Place the stripe tubes in a chilled cold block and the labeled 1.5 mL Eppendorf tube on ice until ready
to use.
1.
Chapter 6 | OncoScan® FFPE Assay Kit Protocol
3.
41
To the tube labeled G, using the column appropriate to the number of reactions add the reagents in
Table 6.11 in the order shown. Pipet up and down 3 times to rinse tips.
NOTE: Gap Fill Mix contain 50% glycerol, so pipet slowly.
Vortex between additions. Vortex the entire Gap Fill Mix for 3 sec at maximum speed. Spin down
briefly in microfuge.
5. Using the column appropriate to the number of reactions, aliquot the specific volumes of the Gap Fill
Master Mix from Table 6.11 to a 12-tube strip. Cover the strip tube with cap. Spin down the strip
tube and place on a chilled cold block until ready to use.
4.
NOTE: In case user has a 8-strip tube microfuge, user can cut 12-tube strip accordingly to
fit 8-tube microfuge.
Table 6.11 Gap Fill Master Mix
Reagent
P/N
1 Reaction
7 Reactions
9 Reactions
13 Reactions
25 Reactions
(~20% overage)
(~20% overage)
(~20% overage)
(~20% overage)
Water
902253
10.58 μL
89 μL
114 μL
165 μL
318 μL
Buffer A
902246
1.18 μL
9.9 μL
12.7 μL
18.3 μL
35.3 μL
SAP, Recombinant (1U/μL)
902251
0.84 μL
7.1 μL
9.1 μL
13.1 μL
25.2 μL
Gap Fill Enzyme Mix
902252
1.40 μL
11.8 μL
15.1 μL
21.8 μL
42.0 μL
14.0 μL
117.6 μL
151.2 μL
218.4 μL
420 μL
16.0 μL
7-Tube Strip
16.0 μL
9-Tube Strip
16.5 μL
12-Tube Strip
33 μL
12-Tube Strip
Total
Volume per Tube for Tube Strip
Add Gap Fill Mix
CAUTION: Make sure the plate is in the correct orientation (well A1 at the top left position)
before pipetting the reagents from the strip tube to the plate.
To Add the Gap Fill Mix:
1.
2.
3.
4.
5.
Remove plate ANNEAL from the thermal cycler, place in a cold block on ice for 1 min; as soon as
you put the plate on ice, stop the OncoScan Anneal program and start the OncoScan Gap Fill program
on the thermal cycler and wait for the thermal cycler to reach 58°C, then pause the program.
After the 1 min incubation on ice, spin the ANNEAL Plate at 2400 rpm for 60 sec and return it to
the cold block.
Slowly and carefully remove the adhesive file in order to avoid any contamination through sample
splashing.
Using a 12-channel P20 pipette, add 14.0 μL Gap Fill Mix to each reaction in the appropriate rows,
as shown in Figure 6.4 below. Pipet up and down 3 times to rinse tips.
 For the 13 or 25 Sample Reaction, add 14 μL of Gap-Fill Master Mix from the G Master Mix Tube
to the Negative Control sample.
Tightly seal the plate, vortex at high speed for 5 sec, then spin down the Anneal Plate at 2400 rpm
for 60 sec. Keep the Anneal Plate on the cold block. The current volume in the Anneal Plate is now
24.0 μL.
Chapter 6 | OncoScan® FFPE Assay Kit Protocol
42
Figure 6.4 Adding Gap Fill Mix to ANNEAL PLATE
7 Samples
10 μL + 14 μL = 24 μL
A
1
2
3
4
5
+ _
6
7
8
9
13 Samples
10 μL + 14 μL = 24 μL
10
11
ANN
12
A
B
B
C
C
D
D
E
E
F
F
G
G
H
H
1
2
1
2
3
4
5
6
7
+ _
8
9
10
11
ANN
12
B
C
D
D
E
E
F
F
G
G
H
H
= Sample + Gap Fill Mix
1
A
C
= Negative Control
5
6
7
8
9
10
11
ANN
12
+
25 Samples
10 μL + 14 μL = 24 μL
B
= Positive Control
4
_
9 Samples
10 μL + 14 μL = 24 μL
A
3
2
3
4
_
= Empty well
5
6
7
8
9
10
11
ANN
12
+
= Master Mix tube
Chapter 6 | OncoScan® FFPE Assay Kit Protocol
43
Channel Split
CAUTION: When transferring sample volumes from one 96-well plate to another 96-well
plate, make sure BOTH plates are at the correct orientation (well A1 at top left) before
pipetting.
1.
Label a fresh 96-well half-skirt PCR plate 1st PCR, clearly marking the alternating AT and GC rows
with blue and red colored markers respectively for each row of sample used. (Label rows A & E as “AT”
with a Blue marker and Rows C & G as “GC” with a Red marker (see Figure 6.6). Mark the reaction wells
in the same colors).
 The samples from the A row on the ANNEAL Plate will be split into the A and C rows on the
1st PCR Plate.
 The samples from the E row on the ANNEAL Plate will be split into rows E and G on the
1st PCR Plate.
NOTE: For samples less than or equal to 12 on the anneal plate, it will be split into two
rows on the Assay Plate for AT and GC in row A & C respectively, as shown in Figure 6.6.
Using a 12-channel P20 pipette, transfer 10.0 μL each reaction from the ANNEAL Plate to the 1st
PCR Plate, as shown in Figure 6.5 and Figure 6.6.
 For the 13 Sample Reaction, add 10 μL of the Negative Control sample into well B01 & 10 μL
into well D01.
 For the 25 Sample Reaction, add 10 μL of the Negative Control sample into well F01 & 10 μL into
well H01.
3. Tightly seal the 1st PCR Plate; then spin down at 2400 rpm for 60 sec.
4. Load the plate on the thermal cycler and resume the program to run (Figure 6.7 on page 45).
2.
Figure 6.5
Chapter 6 | OncoScan® FFPE Assay Kit Protocol
Figure 6.6 Preparing the Gap Fill - 1st PCR Plate
7 Samples
1
A
2
3
4
+ _
5
6
7
8
9 Samples
9
10
11
ANN
12
1
A
B
B
C
C
D
D
E
E
F
F
G
G
H
2
3
4
5
1
A
2
3
4
5
B
+ _
6
7
8
9
- 1st 12
PCR
10 GF11
AT
1
A
2
3
4
GC
E
E
F
F
G
G
H
H
3
4
5
6
7
8
_
10
11
ANN
12
+
1
A
C
D
E
E
F
F
G
G
H
H
2
3
4
5
1
_
_
7
+ _
8
9
11
ANN
12
- 1st 12
PCR
10 GF11
AT
GC
6
7
8
9
10
11
ANN
12
+
_
10 μL
10 μL
D
10
B
D
C
9
25 Samples
9
C
B
8
+ _
13 Samples
2
6
C
D
1
5
B
D
A
+ _
10 μL
+ _
C
B
7
H
10 μL
A
6
2
3
4
5
6
7
8
9
- 1st12
PCR
10 GF11
+ AT
1
A
2
3
4
5
6
7
8
9
- 1st 12
PCR
10 GF11
AT
B
+ GC
C
GC
D
E
E
F
F
G
G
H
H
_
_
+ AT
+ GC
44
Chapter 6 | OncoScan® FFPE Assay Kit Protocol
Figure 6.7 OncoScan Gap Fill Thermal Cycler Program (for GeneAmp® PCR System 9700)
Pause the cycler at
the dotted lines
95°C
Remove the plate at the end
of 58°C and then allow the
cycler to ramp down to 37°C
58°C
95°C
15 min
10 min
58°C
11 min 11 min
37°C
37°C
20 min
15 min
4°C
∞
+4 μL
+ 3 μL
dNTP mix (A/T) Exo mix
or
(well 17.0 μL)
dNTP mix (G/C)
+ 25 μL
Cleavage Mix
(well 42.0 μL)
Blue arrows indicate reagent addition steps
(well 14.0 μL)
Figure 6.8 OncoScan Gap Fill Thermal Cycler Program (for Veriti Thermal Cycler)
Pause the cycler at
the dotted lines to add the
reagents for the next step
95°C
Remove the plate at the end
of 58°C and then allow the
cycler to ramp down to 37°C
95°C
15 min
10 min
58°C
22 min
37°C
37°C
20 min
15 min
4°C
∞
+4 μL
+ 3 μL
dNTP mix (A/T) Exo mix
or
(well 17.0 μL)
dNTP mix (G/C)
+ 25 μL
Cleavage Mix
(well 42.0 μL)
Blue arrows indicate reagent addition steps
45
Chapter 6 | OncoScan® FFPE Assay Kit Protocol
46
NOTE: For the Veriti Thermal Cycler, when programming the “OncoScan Gap Fill” program,
combine the first two 58°C incubations for 11 min each into one stage at 58°C for 22 min (see
Table B.2 on page 105 and Figure B.5 on page 106). This change is recommended because it
has been noticed that the touch screen does not consistently respond to the “PAUSE RUN”
function when paused between the two stages of 58°C. (The touch screen may not pause at
the end of the 58°C at 11 min and will move on to the next 58°C stage). This will ruin the Gap
Fill reaction, leading to assay failure. Using a laboratory timer is critical during the first two
58°C incubations. Make sure the program pauses when the “PAUSE RUN” is touched.
NOTE: It is strongly recommended to use a timer during this stage of the OncoScan® FFPE
Assay Protocol so as not to miss pausing the thermal cycler or important reagent additions.
Add the dNTP AT Mix and dNTP GC Mix
To Add the AT Mix and GC Mix to the Assay Plate:
1.
2.
3.
4.
5.
6.
After 11 min at 58°C, press Pause on the thermal cycler and remove the 1st PCR Plate.
Place the plate in a cold block on ice for 1 min.
Spin down at 2400 rpm for 30 sec. Remove the 1st PCR Plate from the centrifuge and return the plate
to the cold block on ice.
Ensure the plate is at the correct orientation (Well A1 at top left). Carefully remove the adhesive film
in a slow motion to avoid any contamination between samples in wells.
Using a 12-channel P20 pipette, add 4 μL of AT Mix to:
 row A on the 1st PCR Plate for 7, 9 and 13 reactions. Pipet up and down 3 times to rinse tips
(Figure 6.9).
 For the 13 Sample Reaction, add 4 μL of AT Mix from the Master Mix tube to the well B01
on the 1st PCR Plate (Figure 6.9).
 rows A and E on the 1st PCR Plate for 25 reactions. Pipet up and down 3 times to rinse tips
(Figure 6.9).
 For the 25th sample reaction, add 4 μL of the AT Mix from the Master Mix tube to the well
F01 on the 1st PCR Plate.
Using a 12-channel P20 pipette, add 4 μL of GC Mix to:
 row C on the 1st PCR Plate for 7, 9 and 13 reactions. Pipet up and down 3 times to rinse tips
(Figure 6.9).
 For the 13 Sample Reaction, add 4 μL of GC Mix from the Master Mix tube to the well D01
on the 1st PCR Plate (Figure 6.9).
 rows C and G on the 1st PCR Plate for 25 reactions. Pipet up and down 3 times to rinse tips.
 For the 25 Sample Reaction, add 4 μL of GC Mix from the Master Mix tube to the well H01
on the 1st PCR Plate (Figure 6.9).
Chapter 6 | OncoScan® FFPE Assay Kit Protocol
47
Figure 6.9 Adding the dNTP AT Mix and dNTP GC Mix
AT
AT
7 Samples
10 μL + 4 μL = 14 μL
GC
A
1
2
3
4
5
B
+ _
6
7
8
9
- 1st 12
PCR
10 GF11
AT
AT
C
GC
GC
D
D
E
E
F
F
G
GC
H
AT
9 Samples
10 μL + 4 μL = 14 μL
A
B
GC
A
B
+ _
C
13 Samples
10 μL + 4 μL = 14 μL
C
1
2
3
4
5
6
7
+ _
8
+ _
9
F
2
3
- 1st 12
PCR
10 GF11
AT
AT
4
5
6
7
8
- 1st12
PCR
10 GF11
9
+ AT
+ GC
_
G
H
25 Samples
10 μL + 4 μL = 14 μL
AT
1
A
2
3
4
5
6
7
8
9
- 1st 12
PCR
10 GF11
AT
B
GC
C
D
E
1
_
GC
D
GC
E
F
G
G
H
H
+ AT
_
+ GC
_
GC
= Master Mix tube
NOTE: The current volume in the 1st PCR Plate for each sample in the wells is 14 μL.
Tightly seal the plate, vortex at high speed for 5 sec; then spin down at 2400 rpm for 60 sec.
8. Place the 1st PCR Plate back on the thermal cycler and press Resume.
9. Set a timer for 10 min to be back in the lab to pause the thermal cycler at 58°C immediately after the
11 min incubation.
7.
NOTE: During the 58°C incubation for 11 min, prepare the Exo mix.
NOTE: It is CRITICAL to remove the assay plate at 58°C, BEFORE the cycler ramps down to 37°C
to avoid possible non-specific Gapfill.
Chapter 6 | OncoScan® FFPE Assay Kit Protocol
48
Prepare and Add the Exo Mix
NOTE: Starting from this step onward until the end of the assay, the number of reactions will
double due to AT & GC Channel split. Hence all of the master mixes, from the addition of Exo
Mix and beyond, will be prepared to account for twice the number of reactions. The reactions
must be carried out in the same plate layout until the end of the assay.

7 anneal reactions (6 samples including a positive control + 1 Negative control) will be
processed as 14 reactions.

9 anneal reactions (8 samples including a positive control + 1 Negative control) will be
processed as 18 reactions.

13 anneal reactions (12 samples including a positive control + 1 Negative control) will be
processed as 26 reactions.

25 anneal reactions (24 samples including a positive control + 1 Negative control) will be
processed as 50 reactions.
To Prepare and Add the Exo Mix:
1.
2.
3.
4.
5.
6.
Use a bench top cooler to carry the Exo Mix from the –20°C freezer. Vortex for 1 second and spin
down. Place back in cooler.
Label one set of strip tubes with the Letter E.
Aliquot the appropriate volume of the Exo mix directly into each strip tube.
 For 7, 9 or 13 sample reaction, aliquot 7.5 μL of the Exo mix directly into the appropriate number
of wells in the strip tube.
 For 25 sample reaction, aliquot 16.0 μL of the Exo mix directly into each well of the 12-strip tube.
Cap the strip tubes and spin down to remove any bubbles.
Place the strip tubes in a cold block on ice.
To add the Exo Mix to the Assay Plate:
A. When the thermal cycler temperature reaches the end of the 58°C incubation, press Pause and
remove the 1st PCR Plate.
NOTE: Remove the assay plate at 58°C, BEFORE the cycler ramps down to 37°C to avoid
possible non-specific Gapfill.
B. Place the plate in a cold block on ice for 1 min.
C. Resume the program without a plate in the thermal cycler and allow it to ramp down to start of
the 37°C incubation. Pause thermal cycler at 37°C.
D. After 1 min on ice, spin down at 2400 rpm for 30 sec. Remove the plate from the centrifuge and
place the 1st PCR Plate back on the cold block on ice.
E. Ensure the plate is at the correct orientation (Well A1 at top left). Carefully remove the adhesive
film.
F. Using a 12-channel P20 pipette, add 3 μL Exo Mix to each reaction. Pipet up and down 3 times
to rinse tips (Figure 6.10).
 For the 13 or 25 Sample Reaction, add 3.0 μL of Exo Mix reagent to each of the Negative
Control wells (Figure 6.10).
NOTE: The current volume in the Assay Plate is 17 μL per well.
Chapter 6 | OncoScan® FFPE Assay Kit Protocol
49
Figure 6.10 Adding Exo Mix to Plate
7 Samples
A
1
2
3
4
5
B
+ _
6
7
13 Samples
8
9
- 1st 12
PCR
10 GF11
AT
B
+ _
C
A
GC
C
D
D
E
E
F
F
G
G
H
H
1
2
3
4
B
C
1
2
3
4
5
6
7
+ _
8
+ _
6
7
8
9
- 1st12
PCR
10 GF11
+ AT
+ GC
_
9 Samples
A
5
_
25 Samples
9
10
GF11- 1st 12
PCR
AT
1
A
2
3
4
5
6
7
8
9
- 1st 12
PCR
10 GF11
AT
B
GC
C
D
D
E
E
F
F
G
G
H
H
GC
+ AT
_
+ GC
_
= Master Mix tube
Tightly seal the plate, vortex at high speed for 5 sec; then spin down at 2400 rpm for 60 sec.
8. Place the 1st PCR Plate back on the thermal cycler and press Resume.
7.
Prepare and Add the Cleavage Master Mix
To Prepare and Add the Cleavage Master Mix:
Label a set of strip tubes with CM and place in a cold block until ready to use. Label a 1.5 mL
Eppendorf tube as CM and place on ice until ready to use.
2. During the last 5 min at 95°C on the thermal cycler, prepare the Cleavage Master Mix as follows:
3. Use a bench top cooler to carry the Cleavage Enzyme from the –20°C freezer.
1.
A. Vortex (at max speed for 3 seconds) and spin down the Cleavage Buffer that has been sitting on
ice. Return to ice.
B. Vortex the Cleavage Enzyme for 1 sec. Spin down the Cleavage Enzyme and keep on ice.
C. Add the Cleavage Buffer and Cleavage Enzyme needed for the appropriate number of samples
according to the Table 6.12 to the 1.5 mL (or 2.0 mL) Eppendorf tube (CM).
Chapter 6 | OncoScan® FFPE Assay Kit Protocol
50
D. Pipet up and down 3 times to rinse tips. Vortex the CM tube for 3 seconds (3 times - 1 second at
a time) at maximum speed and spin down. Keep on ice.
E. Using the column appropriate to the number of reactions, aliquot the specific volumes of the
Cleavage Master Mix from Table 6.12 to the strip tube labeled CM. Cover the strip tube with cap.
Spin down the strip tube and place on a chilled cold block until ready to use.
NOTE: The master mix tables from Table 6.12 onward give the volumes needed for the
number of reactions as opposed to the number of samples given in previous tables. Since
there are now 2 reactions for every 1 sample.
Table 6.12 Cleavage Master Mix
Number of Runs per Assay Kit
4
3
2
1
Number of Samples (includes 1 positive control per run)
6
8
12
24
Negative Control per Run
1
1
1
1
x 2 Channels
x 2 Channels
x 2 Channels
x 2 Channels
AT & GC Channels
Reagent
P/N
1 Reaction
14 Reactions
18 Reactions
26 Reactions
50 Reactions
(~25% overage)
(~25% overage)
(~25% overage)
(~25% overage)
Cleavage Buffer
902257
25.0 μL
438 μL
563 μL
813 μL
1563 μL
Cleavage Enzyme
902258
0.2 μL
3.5 μL
4.5 μL
6.5 μL
12.5 μL
25.2 μL
441.0 μL
567.0 μL
819.0 μL
1575.0 μL
60.0 μL
7-Tube Strip
60.0 μL
9-Tube Strip
60.0 μL
12-Tube Strip
120.0 μL
12-Tube Strip
Total
Volume per Tube for Tube Strip
4.
To add the Cleavage Master Mix to the Assay Plate:
A. When the thermal cycler temperature reaches the start of the 37°C step, press Pause and remove
the 1st PCR Plate.
B. Place the 1st PCR Plate in a cold block on ice for 1 min.
C. Spin down at 2400 rpm for 30 sec. Remove the Assay Plate from the centrifuge and place it on
the cold block. Carefully remove the adhesive seal from the plate.
D. Using a 12-channel P200 pipette, add 25 μL Cleavage Master Mix to each reaction. Pipet up and
down 3 times to rinse tips (Figure 6.11).
 For the 13 or 25 Sample Reaction, add 25 μL of Cleavage Mix from the Master Mix tube to
each of the Negative Control wells (Figure 6.11).
NOTE: The current volume in the Assay Plate is 42.0 μL.
Tightly seal the plate, vortex at high speed for 5 sec; then spin down at 2400 rpm for 60 sec.
6. Place the 1st PCR plate back on the thermal cycler and press Resume.
7. Set a timer for 25 min to leave 5 min to make the master mix for the next PCR step.
5.
Chapter 6 | OncoScan® FFPE Assay Kit Protocol
51
Figure 6.11 Adding Cleavage Master Mix to Plate
7 Samples
A
1
2
3
4
5
B
+ _
6
7
13 Samples
8
9
- 1st 12
PCR
10 GF11
AT
B
+ _
C
A
GC
C
D
D
E
E
F
F
G
G
H
H
1
2
3
4
1
2
3
4
5
6
B
7
+ _
8
+ _
C
6
7
8
9
- 1st12
PCR
10 GF11
+ AT
+ GC
_
9 Samples
A
5
_
25 Samples
9
10
GF11- 1st 12
PCR
AT
1
A
2
3
4
5
6
7
8
9
- 1st 12
PCR
10 GF11
AT
B
GC
C
D
D
E
E
F
F
G
G
H
H
GC
_
_
+ AT
+ GC
= Master Mix tube
Prepare and Add the PCR Mix
Label one set of strip tubes PCR and place in a cold block until ready to use. Label a 1.5 mL
Eppendorf tube (or 2.0 mL Eppendorf tube for 50 reactions) as PCR and place on ice until ready to
use.
2. During the last 5 min at 95°C on the thermal cycler, prepare the PCR Mix as follows:
1.
A. Using a bench top cooler, carry the Taq Polymerase from the –20°C freezer.
B. Vortex and spin down the PCR Mix that has been sitting on ice.
Vortex the Taq Polymerase for 1 sec and spin down. Return to ice.
C. Add the PCR Mix and Taq Enzyme needed for the appropriate number of samples according to
the Table 6.13 to the 1.5 mL (or 2.0 mL) Eppendorf tube labeled as PCR. Pipet up and down
3 times to rinse tips. Vortex the PCR Master Mix tube for 3 seconds (3 times - 1 second at a time)
at maximum speed and spin down. Keep on ice.
D. Using the column appropriate to the number of reactions, aliquot the specific volumes of the
1st PCR Master Mix from Table 6.13 to the strip tube labeled PCR. Cover the strip tube with
caps. Spin down the strip tube and place on a chilled cold block until ready to use.
Chapter 6 | OncoScan® FFPE Assay Kit Protocol
52
Table 6.13 1st PCR Master Mix
Number of Runs per Assay Kit
4
3
2
1
Number of Samples (includes 1 positive control per run)
6
8
12
24
Negative Control per Run
1
1
1
1
x 2 Channels
x 2 Channels
x 2 Channels
x 2 Channels
AT & GC Channels
Reagent
P/N
1 Reaction
14 Reactions
18 Reactions
26 Reactions
50 Reactions
(~25% overage)
(~25% overage)
(~25% overage)
(~25% overage)
PCR Mix
902259
24.4 μL
428 μL
550 μL
794 μL
1528 μL
Taq Polymerase
902260
0.56 μL
9.8 μL
12.6 μL
18.1 μL
34.9 μL
25.0 μL
437 μL
562 μL
812 μL
1562 μL
60.0 μL
7-Tube Strip
60.0 μL
9-Tube Strip
60.0 μL
12-Tube Strip
120.0 μL
12-Tube Strip
Total
Volume per Tube for Tube Strip
Figure 6.12 OncoScan 1st PCR Thermal Cycler Program
95°C
95°C
1 min 20s
72°C
60°C
60°C
30s
10s
10s
72°C
5 min
20 cycles
4°C
∞
Once the OncoScan GAP Fill program completes, remove the 1st PCR plate and place on a chilled cold
block for 1 min. After the 1 min incubation, make sure the seal is tight on the plate, then vortex and
spin down the 1st PCR plate for 30 sec.
4. Stop the OncoScan GAP Fill program and start the OncoScan 1st PCR program, pause program when
thermal cycler reaches 60°C.
5. Add the PCR mix to the Assay plate and remember to carefully remove the adhesive. Using a 12channel P200 pipette, add 25 μL PCR Master Mix to each reaction. Pipet up and down 3 times to
rinse tip (Figure 6.13).
 For the 13 or 25 Sample Reaction, add 25 μL of the PCR master Mix reagent from the Master Mix
tube to each of the Negative Control wells (Figure 6.13).
3.
NOTE: The currently volume in the Assay Plate is 67.0 μL.
Chapter 6 | OncoScan® FFPE Assay Kit Protocol
53
Figure 6.13 Adding 1st PCR Master Mix to Plate
7 Samples
A
1
2
3
4
5
B
+ _
6
7
13 Samples
8
9
- 1st 12
PCR
10 GF11
AT
B
+ _
C
A
GC
C
D
D
E
E
F
F
G
G
H
H
1
2
3
4
B
C
1
2
3
4
5
6
7
+ _
8
+ _
6
7
8
9
- 1st12
PCR
10 GF11
+ AT
+ GC
_
9 Samples
A
5
_
25 Samples
9
10
GF11- 1st 12
PCR
AT
1
A
2
3
4
5
6
7
8
9
- 1st 12
PCR
10 GF11
AT
B
GC
C
D
D
E
E
F
F
G
G
H
H
GC
_
_
+ AT
+ GC
= Master Mix tube
Tightly seal the plate, vortex at high speed for 5 sec; then spin down at 2400 rpm for 60 sec.
7. Place the plate on the thermal cycler and press Resume.
8. When the program has ended, transfer the sealed 1st PCR Plate to the Post-PCR Lab and place on
ice.
6.
IMPORTANT: To prevent contamination from PCR products, the Assay Plate must remain
tightly sealed until it has been transferred to the Post-PCR Lab. DO NOT open the seal of
the Assay Plate after PCR or spin down in the Pre-PCR Lab.
Chapter 6 | OncoScan® FFPE Assay Kit Protocol
54
Stage 3 — First QC Gel and 2nd PCR (Post-PCR Lab)
About this Stage
This stage begins in the Post-PCR Lab. During this stage, the sealed 1st PCR Plate will be transferred
to the Post-PCR Lab. A 2nd PCR amplification will be set up and run in the Post-PCR Lab. An aliquot
from each sample of the 1st PCR Plate will be taken and a QC gel will be performed to check the
amplified products.
NOTE: Make sure all three required thermal cycler programs (OncoScan 2nd PCR, OncoScan
HaeIII, OncoScan Hybridization) are set up on the thermal cycler BEFORE starting this stage.
Refer to Appendix B for programs and detailed instructions for programming the Veriti
Thermal Cyclers.
Location and Duration



Post-PCR Lab
Hands-on time: 20 min
Thermal cycler time: 25 min
Input Required from Previous Stage
Table 6.14
Item
1st PCR Plate with amplified DNA from the Pre-PCR Lab
Equipment and Materials Required
Table 6.15 Equipment and Materials Required for Stage 3 — First QC Gel and 2nd PCR (Post-PCR Lab)
Quantity
Item
3
Cold block, chilled in 4°C refrigerator
1
Centrifuge, plate
1
Ice container, rectangular, filled with ice
As required
MicroAmp Clear Adhesive Films
As required
Eppendorf tube, 2.0 mL, Nuclease-free
As required
Eppendorf tube, 1.5 mL, Nuclease-free
1
Microcentrifuge for tubes
1
Microcentrifuge for strip tubes
1 each
Pipettes:
Single-channel P200
 Single-channel P1000
 12-channel P20

As required
1
Pipette tips
Thermal Cycler, 96-well GeneAmp PCR System 9700 (gold or silver block), Veriti Thermal Cycler
Chapter 6 | OncoScan® FFPE Assay Kit Protocol
55
Table 6.15 Equipment and Materials Required for Stage 3 — First QC Gel and 2nd PCR (Post-PCR Lab) (Continued)
Quantity
Item
1
Tube strips with caps, PCR 12-well
2
Plate, 96-well half-skirt PCR
1
Gel Box (Wide-Mini sub cell GT basic system)
1
Power Supply for Gel Electrophoresis
1
AlphaImager HP System for gel imaging (with software)
1
Bench top cooler
1
Blue Permanent Marker, Extra fine tip
1
Red Permanent Marker, Extra fine tip
1
Compression Pad
1
Timer
OncoScan® Reagent Components Required
Table 6.16 OncoScan® Reagent Components Required
OncoScan Reagents from Module OncoScan 2nd Stage PCR and Post PCR Processing
PCR Mix
Taq Polymerase
QC Gel Materials Required
Table 6.17 QC Gel Materials Required (Not included in the OncoScan® FFPE Assay Kit)
Quantity
Item
Vendor and Part Number
1
NEB Low Molecular Weight Ladder
New England Biolabs, P/N N3236S
1
Gel loading Dye, Blue (6X)
New England Biolabs, P/N B7021S
1
100% Glycerol
Affymetrix, P/N 16374
1
EDTA, 0.5 M solution
Affymetrix, P/N 15694
1
3% Agarose gel
Bio-Rad Precast ReadyAgarose™ Wide-Mini Gel,
P/N 161-3040
Transfer Assay Plate to Post-PCR Lab
If you have not already done so, transfer 1st PCR Plate to the Post-Amp Lab and place on ice.
IMPORTANT: To prevent contamination from PCR products, the 1st PCR Plate must remain
tightly sealed until it has been transferred to the Post-Amp Lab.
Chapter 6 | OncoScan® FFPE Assay Kit Protocol
56
Preparing the 1st PCR Plate for the 2nd PCR Stage
1.
Ensure the seal is tight. Vortex the 1st PCR Plate at max speed for 5 sec. Spin down the plate at
2400 rpm for 60 sec. Keep on cold block until ready to use.
Prepare the 2nd PCR Mix and Setup 2nd PCR
CAUTION: When transferring sample volumes from one 96-well plate to another 96-well
plate, make sure BOTH plates are at the correct orientation (well A1 at top left) before
pipetting.
1.
2.
3.
4.
5.
6.
7.
Turn on thermal cycler to heat lid.
Remove the PCR mix from the –20°C freezer and thaw at room temperature for 10-15 minutes.
Fill a rectangular ice bucket with ice and place 3 chilled cold blocks on the ice.
Label a new 96-well half-skirt PCR plate as 2nd stage PCR and keep in a cold block on ice. Following
the same layout as the 1st PCR Plate, label row A (or A & E for 25 reactions) as “AT” with a Blue marker
and Row C (or Rows C & G for 25 reactions) as “GC” with a Red marker. Mark the reaction wells in the
same colors. Cover the plate with a plate seal until ready for use.
Label a strip tube and a 1.5 mL Eppendorf tube as PCR 2. Place the labeled strip tube in a chilled
cold block and the 1.5 mL tube on ice until ready to use.
Start the OncoScan 2nd PCR program and pause when the thermal cycler reaches 60°C.
Prepare the PCR Mix as follows:
A. Use a bench top cooler to carry the Taq Polymerase from the –20°C freezer.
B. Once thawed, vortex and spin down the PCR Mix. Keep on ice.
C. Vortex the Taq Polymerase for 1 sec. Spin down the Taq Polymerase. Keep on ice.
D. Add the PCR Mix and Taq Polymerase as needed for the appropriate number of samples
according to the Table 6.18 to the 1.5 mL Eppendorf tube (PCR 2). Pipet up and down 3 times
to rinse tips. Vortex the PCR Master Mix tube for 3 seconds (3 times - 1 second at a time) at
maximum speed and spin down. Keep on ice.
E. Using the column appropriate to the number of reactions, aliquot the specific volumes of the
2nd PCR Master Mix from Table 6.18 to a 12-tube strip. Cover the strip tube with cap. Spin
down the strip tube and place on a chilled cold block until ready to use.
F. Using a 12-channel P200 pipette, add 25 μL 2nd PCR Master Mix from the strip tube to the
appropriate wells in the new 2nd PCR plate (Figure 6.14).
 For the 13 or 25 Sample Reaction, add 25 μL of the 2nd PCR Master Mix from the Master Mix
tube to each of the Negative Control wells (Figure 6.14).
Chapter 6 | OncoScan® FFPE Assay Kit Protocol
57
Figure 6.14 2nd Stage PCR
7 Samples
A
1
2
3
4
5
B
+ _
6
7
13 Samples
8
9
2nd
PCR
10 stage
11
12
AT
B
+ _
C
A
C
GC
D
D
E
E
F
F
G
G
H
H
1
2
3
4
1
2
3
4
5
6
7
B
+ _
8
7
8
9
2nd
PCR
10 stage
11
12
AT
+
+ GC
25 Samples
9
2nd
PCR
10 stage
11
12
AT
1
A
2
3
4
5
6
7
8
9
2nd
PCR
10 stage
11
12
AT
B
+ _
C
6
_
9 Samples
A
5
_
C
GC
D
D
E
E
F
F
G
G
H
H
GC
+ AT
_
+ GC
_
= Master Mix tube
Table 6.18 2nd PCR Master Mix
Number of Runs per Assay Kit
4
3
2
1
Number of Samples (includes 1 positive control per run)
6
8
12
24
Negative Control per Run
1
1
1
1
x 2 Channels
x 2 Channels
x 2 Channels
x 2 Channels
AT & GC Channels
Reagent
P/N
1 Reaction
14 Reactions
18 Reactions
26 Reactions
50 Reactions
(~25% overage)
(~25% overage)
(~25% overage)
(~25% overage)
PCR Mix
902259
24.4 μL
428 μL
550 μL
794 μL
1528 μL
Taq Polymerase
902260
0.56 μL
9.8 μL
12.6 μL
18.1 μL
34.9 μL
25.0 μL
437 μL
562 μL
812 μL
1562 μL
60.0 μL
7-Tube Strip
60.0 μL
9-Tube Strip
60.0 μL
12-Tube Strip
120.0 μL
12-Tube Strip
Total
Volume per Tube for Tube Strip
Chapter 6 | OncoScan® FFPE Assay Kit Protocol
8.
58
Remove the seal from the 1st PCR Plate carefully and add 2 μL from each well to the corresponding
wells in the 2nd PCR Plate. Pipet mix 3 times up and down to rinse pipette tips. Cover the 1st PCR
Plate loosely with a seal until ready to use for QC gel procedure. Keep 1st PCR Plate on a chilled cold
block (Figure 6.15).
 For the 13 or 25 Sample Reaction, add 2 μL of the Negative Control 1st PCR product to the
appropriate negative control wells (Figure 6.15).
Figure 6.15
7 Samples
A
1
2
3
4
5
B
+ _
6
7
13 Samples
8
9
GF11- 1st 12
PCR
AT
A
B
+ _
C
10
GC
C
D
D
E
E
F
F
G
G
H
H
1
2
3
4
1
2
3
4
5
+ _
6
7
8
9
2nd
stage
PCR
10
11
12
AT
A
B
+ _
C
GC
C
D
D
E
E
F
F
G
G
H
H
2
3
4
5
6
7
B
+ _
8
9
- 1st 12
PCR
10 GF11
AT
1
2
3
4
GC
E
F
F
G
G
H
H
B
C
7
8
+ AT
2nd
PCR
10 stage
11
12
AT
9
+
+ GC
_
1
2
3
4
5
6
7
8
9
- 1st 12
PCR
10 GF11
AT
GC
+ AT
_
+ GC
_
2 μL
4
6
C
E
3
- 1st12
PCR
10 GF11
B
D
2
5
_
A
D
1
9
25 Samples
+ _
C
A
8
+ GC
9 Samples
1
7
2 μL
B
A
6
_
2 μL
A
5
_
2 μL
5
6
7
+ _
8
+ _
9
2nd
stage
PCR
10
11
12
AT
1
A
2
3
4
5
6
7
8
9
2nd
PCR
10 stage
11
12
AT
B
GC
C
D
D
E
E
F
F
G
G
H
H
GC
_
_
+ AT
+ GC
Chapter 6 | OncoScan® FFPE Assay Kit Protocol
59
Seal the 2nd PCR Plate. Vortex for 5 sec at max speed and spin down at 2400 rpm for 60 seconds.
10. Place the 2nd PCR Plate on the thermal cycler and run the OncoScan 2nd PCR program (Figure 6.16)
9.
Figure 6.16 OncoScan 2nd PCR Thermal Cycler Program
95°C
95°C
1 min 20s
72°C
60°C
60°C
30s
10s
10s
72°C
5 min
15 cycles
4°C
∞
Prepare and Run the First QC Gel
The first quality control gel is used to check for first PCR product.
NOTE: The instructions below are specific for running the First PCR QC gel on 3% Agarose gel
and electrophoresis chamber mentioned in Table 6.25. Adjust the gel running conditions
based on the Gel chamber used to run the gels. Run gels at 5V/cm (5 volts × Distance in cm
between electrodes). For example, run a 33 cm electrophoresis box at 165V; run a 16 cm
electrophoresis box at 80V. Please refer to Appendix D for instructions on running on
4% E-Gels.
Prepare the Gel Materials
While the OncoScan 2nd PCR program is running, prepare the materials required for the first QC gel.
Reagents for Gel Loading
1.
Glycerol-EDTA buffer (50% Glycerol + 50 mM EDTA) (Table 6.19):
Table 6.19 Glycerol-EDTA Buffer (50% Glycerol + 50 mM EDTA)
Reagent
100% Glycerol
0.5 M EDTA
Nuclease-free Water
Total Volume
Initial Concentration
Final Concentration
Volume
100%
50%
1000.0 μL
500 mM
50 mM
200.0 μL
N/A
N/A
800.0 μL
2000.0 μL
Chapter 6 | OncoScan® FFPE Assay Kit Protocol
2.
60
1:10 Dilution of 6X Gel Loading Dye (Table 6.20): (Store at 4°C for long-term storage)
Table 6.20 1:10 Dilution of 6X Gel Loading Dye
Reagent
Volume
Glycerol-EDTA buffer (50% Glycerol + 50 mM EDTA)
900.0 μL
NEB 6X Loading Dye
100.0 μL
Total Volume
3.
1000.0 μL
Dilution of the 50 bp ladder (Table 6.21):
(1 mL of the diluted ladder can be used in ~200 lanes, Store at –20°C for long-term storage)
Table 6.21 Dilution of the 50 bp Ladder
Reagent
Volume
Glycerol-EDTA buffer (50% Glycerol + 50 mM EDTA)
830.0 μL
NEB 6X Loading Dye
100.0 μL
NEB 50 bp Ladder
70.0 μL
Total Volume
1000.0 μL
Prepare the QC Gel 1 Plate
CAUTION: When transferring sample volumes from one 96-well plate to another 96-well
plate, make sure BOTH plates are in the correct orientation (well A1 at top left) before
pipetting.
To Prepare the Gel Plate:
1.
Label one fresh PCR plate Gel1 (the gel plate) and load it as follows:
A. Aliquot 8 μL of 1st PCR Plate product to the appropriate wells.
B. Add 2 μL of 1/10th diluted gel loading dye. Pipet up and down three times to rinse the tips.
2.
3.
4.
5.
6.
7.
Seal 1st PCR Plate tightly and keep in a cold block on ice.
Total volume left in each well is approximately 56.0 μL
Tightly seal the gel QC plate, vortex and spin down.
Load 10 μL of each reaction onto a 3% agarose gel.
Load 5.0 μL of diluted 50 bp ladder on both sides of the gel marked as “M”.
Run the gel at 150V for 15 min.
Examine the gel in a gel imager to ensure PCR products are approximately 120 bp.
Figure 6.17 illustrates good results for each sample.
Chapter 6 | OncoScan® FFPE Assay Kit Protocol
61
Figure 6.17 For samples in which successful amplification has occurred, one single band at approximately120 base
pairs should be seen. No distinct band at approximately 120 base pairs should be visible in the Negative Control and in
samples in which amplification has not occurred.
Figure 6.18 Poor QC Gel for First QC Gel (1st PCR)
Blue Circles:
Red Circles:
QC gel pattern: Faint or no PCR product visible on
the 1st PCR QC gel image in AT or GC channel for a
given sample.
QC gel pattern: Faint or no PCR product visible on the
1st PCR QC gel image in BOTH AT and GC channel for a
given sample.
Possible Causes: Gel loading error or mis-pipetting.
Possible Causes: Poor DNA quality, inaccurate DNA
quantitation or mis-pipetting the DNA or MIP probe.
Solution:


Eliminate the gel loading error by running the QC gel
again.
If the repeat gel shows the same faint/no PCR band in
a given channel, there was likely a mis-pipetting step
after the Channel Split in the Pre-PCR stage. Repeat
this sample from the beginning of the assay.
Solution:


Recheck the quantification for the sample by using the
recommended PicoGreen protocol to make sure you
have 80 ng of starting material.
Mis-pipetting in the Anneal step by not adding the
probe mix or the input DNA can result in no PCR bands.
Repeat Assay from the beginning.
DO NOT hybridize these samples onto the Array.
DO NOT hybridize these samples onto the Array.
(Refer to the Troubleshooting section for details.)
Chapter 6 | OncoScan® FFPE Assay Kit Protocol
62
QC Check Point 1
If any of the samples have the similar pattern shown in the “poor QC” results (Figure 6.18), run the
gel again to make sure there was no gel loading error.
If the repeat gel shows the same pattern, do not proceed with the assay for the problematic samples.
Refer to the troubleshooting guidelines and repeat the assay again. DO NOT hybridize these samples
onto the arrays.
Chapter 6 | OncoScan® FFPE Assay Kit Protocol
Stage 4 — HaeIII Digest and Second QC Gel
About this Stage
During this stage, smaller DNA fragments are generated to improve sample hybridization onto the
OncoScan® Arrays. The DNA fragment size is then checked on the second QC gel.
Location and Duration



Post-PCR Lab
Hands-on time: 30 min
Thermal cycler time: 1 hr 45 min
Input Required from Previous Stage
Table 6.22
Item
2nd PCR Plate with Amplified DNA
Equipment and Materials Required
Table 6.23 Equipment and Materials Required for Stage 4 — HaeIII Digest and Second QC Gel
Quantity
Item
2
Cold blocks, chilled in 4°C refrigerator
1
Centrifuge, plate
1
Ice container, rectangular, filled with ice
As required
MicroAmp Clear Adhesive Films
1
Microcentrifuge for tubes
1
Microcentrifuge for strips
1 each
Pipettes:
Single-channel P200
 Single-channel P1000
 12-channel P20
 12-channel P200
 Optional: 24-channel; P20

As required
Pipette tips
2
Plate, 96-well PCR
1
Thermal Cycler, 96-well GeneAmp PCR System 9700 (gold or silver block), Veriti Thermal Cycler
1
Tube, Eppendorf 1.5 mL
2
Tube strips with caps, PCR 12-well
1
Bench Top Cooler
1
Blue Permanent Marker, Extra fine tip
63
Chapter 6 | OncoScan® FFPE Assay Kit Protocol
64
Table 6.23 Equipment and Materials Required for Stage 4 — HaeIII Digest and Second QC Gel (Continued)
Quantity
Item
1
Red Permanent Marker, Extra fine tip
1
Compression Pad
1
Timer
OncoScan® Reagent Components Required
Table 6.24 OncoScan® Reagent Components Required
OncoScan Reagent from module 2nd Stage PCR and Post PCR Processing
Buffer B
HaeIII Enzyme
Exonuclease I
Gel Materials Required
Table 6.25 Gel Materials Required
Quantity Item
Supplier
1
1X TE Buffer
Affymetrix P/N 75792
1
NEB Low Molecular Weight Ladder, diluted
Diluted 50 bp ladder, prepared as instructed on page 60.
1:10 Loading buffer
1:10 diluted Gel Loading Dye prepared as instructed on
page 60.
3% Agarose gel
Bio-Rad Precast Ready Agarose Wide-Mini Gel,
P/N 161-3040
2 μL/rxn
1
Thaw the Reagents
To Thaw the Reagents:
Place the Buffer B on the bench top for 10-15 minutes to thaw at room temperature.
2. Once thawed, vortex Buffer B at max speed for 3-5 seconds, spin down briefly and place on ice.
1.
Chapter 6 | OncoScan® FFPE Assay Kit Protocol
65
Prepare and Run the HaeIII Digest
To Prepare and Run the HaeIII Digest:
1.
2.
3.
4.
5.
6.
7.
Label one fresh 96-well half-skirt PCR Plate HaeIII. Following the same layout as the 1st PCR Plate,
label row A (or A & E if running 25 reactions) as “AT” with a Blue marker and Row C (or C & G if
running 25 reactions) as “GC” with a Red Marker. Mark the reaction wells in the same colors. Keep it
loosely covered with seal on chilled cold block.
Stop the PCR program after the completion of the 2nd PCR and remove the 2nd PCR Plate from
the thermal cycler. Place the 2nd PCR Plate on chilled cold block for 1 min. After 1 min, ensure the
seal is tight and Vortex for 5 seconds at max speed. Spin down at 2400 rpm for 60 sec. Return the
plate to the cold block until ready to use for the HaeIII digest.
Label a strip tube and a 1.5 mL Eppendorf tube with HaeIII.
Place the labeled strip in a chilled cold block and the labeled 1.5 mL Eppendorf tube on ice.
Using a bench top cooler, carry the Hae III and Exo enzymes from the –20°C. Vortex briefly and spin
down. Place on ice.
Add the Hae III Mix and Exo Enzyme needed for the appropriate number of samples according to
the Table 6.26 to the 1.5 mL Eppendorf tube (Hae III). Pipet up and down 3 times to rinse tips.
Vortex the HaeIII Master Mix tube briefly at maximum speed and spin down. Keep on ice.
Using the column appropriate to the number of reactions, aliquot the specific volumes of the HaeIII
Master Mix from Table 6.26 to a 12-tube strip. Cover the strip tube with cap. Spin down the strip
tube and place on a chilled cold block until ready to use.
Table 6.26 HaeIII Master Mix
Reagent
P/N
1 Reaction
14 Reactions
18 Reactions
26 Reactions
50 Reactions
(~20% overage)
(~20% overage)
(~20% overage)
(~20% overage)
Buffer B
902261
19.10 μL
321 μL
413 μL
596 μL
1146 μL
HaeIII Enzyme
902262
0.40 μL
6.7 μL
8.6 μL
12.5 μL
24.0 μL
Exo Enzyme
902263
0.50 μL
8.4 μL
10.8 μL
15.6 μL
30.0 μL
20.0 μL
336 μL
432 μL
624 μL
1200 μL
45.0 μL
7-Tube Strip
45.0 μL
9-Tube Strip
48.0 μL
12-Tube Strip
96.0 μL
12-Tube Strip
Total
Volume per Tube for Tube Strip
Chapter 6 | OncoScan® FFPE Assay Kit Protocol
66
CAUTION: When transferring sample volumes from one 96-well plate to another 96-well
plate, make sure BOTH plates are at the correct orientation (well A1 at top left) before
pipetting.
8.
Using a 12-channel P20 pipette, add 20 μL of HaeIII Master Mix to corresponding well of the HaeIII
Plate (Figure 6.19).
 For the 13 or 25 Sample Reaction, add 20 μL of the HaeIII Master Mix from the Master Mix tube
to each of the Negative Control wells (Figure 6.19).
Figure 6.19 Adding HaeIII Master Mix to Plate
7 Samples
A
1
2
3
4
5
B
+ _
6
7
13 Samples
8
9
10
11
A
B
+ _
C
HaeIII
12
AT
GC
C
D
D
E
E
F
F
G
G
H
H
1
2
3
4
B
C
1
2
3
4
5
6
7
+ _
8
+ _
6
7
8
9
10
11
_
HaeIII
12
+ AT
+ GC
_
9 Samples
A
5
25 Samples
9
10
11
HaeIII
12
AT
1
A
2
3
4
5
6
7
8
9
10
HaeIII
12
AT
11
B
GC
C
D
D
E
E
F
F
G
G
H
H
GC
_
_
+ AT
+ GC
= Master Mix tube
9.
Using a 12-channel P20 pipette, add 10 μL of 2nd PCR amplified DNA from 96 well plate labeled
2nd stage PCR to the corresponding wells of the HaeIII Plate. Pipet up and down 3 times to rinse
the tips (Figure 6.20).
 For the 13 or 25 Sample Reaction, add 10 μL of the 2nd PCR from the Negative control to each
of the appropriate Negative Control wells (Figure 6.20).
NOTE: Total volume each well: 30 μL.
Chapter 6 | OncoScan® FFPE Assay Kit Protocol
Figure 6.20
7 Samples
A
1
2
3
4
+ _
5
6
B
7
13 Samples
8
9
2nd
stage
PCR
10
11
12
AT
B
+ _
C
A
GC
C
D
D
E
E
F
F
G
G
H
H
1
2
3
4
1
2
3
4
5
B
+ _
6
7
8
9
10
11
HaeIII
12
AT
A
B
GC
C
D
D
E
E
F
F
G
G
H
H
1
2
3
4
5
6
7
B
+ _
8
9
2nd
stage
PCR
10
11
12
AT
+ GC
1
2
3
4
E
F
F
G
G
H
H
B
C
5
6
7
8
9
10
11
HaeIII
12
+ AT
+ GC
_
1
2
3
4
5
6
7
8
9
2nd
PCR
10 stage
11
12
AT
GC
+ AT
_
+ GC
_
10 μL
4
5
C
E
3
+
B
GC
D
2
2nd
PCR
10 stage
11
12
AT
_
A
D
1
9
25 Samples
+ _
C
A
8
_
9 Samples
A
7
10 μL
+ _
C
6
_
10 μL
A
5
10 μL
6
7
+ _
8
+ _
9
10
11
HaeIII
12
AT
1
A
2
3
4
5
6
7
8
9
10
11
HaeIII
12
AT
B
GC
C
D
D
E
E
F
F
G
G
H
H
GC
_
_
10. Seal the HaeIII Plate tightly while the plate is in a chilled cold block.
11. Vortex the plate at high speed for 5 sec, and spin down at 2400 rpm for 60 sec.
12. Place the plate on a thermal cycler and run the OncoScan HaeIII program (Figure 6.21).
+ AT
+ GC
67
Chapter 6 | OncoScan® FFPE Assay Kit Protocol
68
13. Set a timer for 85 min to pause the thermal cycler at 37°C to remove the aliquot for QC gel. The
HaeIII Plate needs to be paused before the 95°C at 10 min in order to observe the size of the DNA
after HaeIII digest. Setting the timer for 85 min will allow enough time to return to the thermal cycler
and pause the program at 37°C at 88 minutes (2 minutes before the program reaches 95°C).
Figure 6.21 HaeIII Thermal Cycler Program
95°C
10 min
37°C
37°C
88 min
2 min
4°C
Pause at 88 min
to remove QC gel
∞
Prepare and Run the HaeIII Gel (Second QC Gel)
Run the second quality control gel to check the HaeIII Digestion reaction.
NOTE: The instructions below are specific for running the HaeIII QC gel on 3% Agarose gel
and electrophoresis chamber mentioned in Table 6.25. Adjust the gel running conditions
based on the Gel chamber used to run the gels. Run gels at 5V/cm (5 volts × Distance in cm
between electrodes). For example, run a 33 cm electrophoresis box at 165V; run a 16 cm
electrophoresis box at 80V. Please refer to Appendix D for instructions on running the HaeIII
gel QC.
To Prepare and Run the Second Quality Control Gel:
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
Label a 96-well PCR plate Gel2 (the gel plate). Ensure the plate is at the correct orientation (well A1
at top left).
Add 4 μL of 1X TE to the gel plate.
Add 2 μL of 1:10 Gel Loading Dye to each reaction, pipetting up and down 3 times to rinse the tips.
When the cycler reaches 88 min at 37°C, pause the cycler.
Remove the HaeIII Plate and place it on a cold block for 1 minute.
Ensure the HaeIII Plate is tightly sealed and vortex at max speed for 5 seconds. Spin down the plate
at 2400 rpm for 60 seconds.
Ensure the plate is at the correct orientation (well A1 at top left). Remove the plate seal slowly and
carefully.
Remove 4 μL of HaeIII digest sample and add it to the Gel 2 Plate.
Seal the HaeIII plate and place it back on the thermal cycler. Resume the OncoScan HaeIII program.
Seal the QC Gel 2 sample plate, Vortex and Spin down briefly.
Load 10 μL of each reaction and the ladder onto a 3% agarose gel.
Load 3.5 μL of diluted 50 bp ladder on both sides of the gel marked as “M”.
Run the gel at 150V for 15 min.
Examine the gel in a gel imager to ensure that a doublet bands around 40-70 bp are observed.
Chapter 6 | OncoScan® FFPE Assay Kit Protocol
69
Figure 6.22 illustrates a good HaeIII digest gel results.
Figure 6.22 The gel indicates both successful amplification during the second stage PCR reaction and HaeIII digestion.
The predominant pattern should be two bands at approximately 40 and 70 bp.
QC Check Point 2
If any of the samples DO NOT have the similar gel banding pattern shown in the “Good QC” results
(Figure 6.22) or have a gel banding pattern similar to the negative control, run the gel again to make sure
there was no gel loading error.
If the repeat gel of a given sample shows a smear similar to that of the negative control, do not proceed
with the assay. DO NOT hybridize these samples onto the arrays.
Refer to the troubleshooting guidelines and repeat the assay again, as mentioned below.
Prepare and set up a new 2nd PCR.
2. Prepare and set up a new HaeIII using the newly setup PCR.
3. Run the HaeIII gel QC again
1.
Chapter 6 | OncoScan® FFPE Assay Kit Protocol
70
Stage 5 — Hybridization
About this Stage
During this stage, each reaction is denatured then loaded onto an OncoScan® Array – one well (either
AT or GC) per array. The arrays are then placed into a hybridization oven that has been preheated to
49°C. Samples are left to hybridize for 16 to 18 hours.
To help ensure the best results, carefully read the information below before you begin this stage.
IMPORTANT:

Make sure the QC check point criteria are met before processing this stage. For a
sample to be hybridized onto the array, BOTH 1st PCR QC gel and HaeIII QC gel must
meet the QC criteria.

Be sure to equilibrate the arrays to room temperature; otherwise, the rubber septa may
crack and the array may leak.

An accurate hybridization temperature is critical for this assay. Therefore, we recommend
that your hybridization oven(s) be serviced at least once per year to ensure that they are
operating within specifications.

If you will be running more than 2 batches through a given Fluidics Stations, we
recommend that you stagger the hybridization of the arrays by 2 hours so that the arrays
in the third or fourth batch will not be in the hyb oven for more than 18 hours.
Location and Duration




Post-PCR Lab
Hands-on time: approximately 1 hr
Thermal cycler time: 15 min
Hybridization time: 16 to 18 hours
Input Required from Previous Stage
Table 6.27
Item
HaeIII Digest Plate containing HaeIII Digested DNA
Equipment and Consumables Required
The following equipment and consumables are required for this stage.
Table 6.28 Equipment and Consumables Required for Stage 5 — Hybridization
Quantity
1
2 per sample
Item
Cold block, chilled to 4°C (do not freeze)
OncoScan® Array
1
GeneChip® Hybridization Oven 645
1
Ice bucket, filled with ice
1
Razor blade
As required
MicroAmp Clear Adhesive Film
Chapter 6 | OncoScan® FFPE Assay Kit Protocol
71
Table 6.28 Equipment and Consumables Required for Stage 5 — Hybridization (Continued)
Quantity
1 each
As required
Item
Pipettes:
 Single channel P200
 Single-channel P1000
 12-channel P200
Pipette tips for pipettes listed above; full racks
1
Plate, 96-well PCR
1
Plate centrifuge
1
Plate holder
1
Reagent Reservoir, 25 mL
1
Thermal Cycler, 96-well GeneAmp PCR System 9700 (gold block), Veriti Thermal Cycler
1
Compression Pad for Thermal Cycler
2 per array
Tough-Spots®, 1/2 in. diameter
OncoScan® Reagent Components Required
Table 6.29 OncoScan® Reagent Components Required
OncoScan Reagents from Module OncoScan 2nd Stage PCR and Post PCR Processing
Hybridization Mix
Water
Preheat the Hybridization Ovens
To Preheat the Hybridization Ovens:
1.
2.
3.
4.
5.
Turn each Hybridization Oven 645 on and press the “Pause” button to start.
Set the temperature to 49°C.
Set the rpm to 60.
Press the run button once to allow hybridization oven to start. Allow to preheat.
Allow approximately 30 minutes to 1 hour for the oven to preheat.
Prepare the Arrays
To Prepare the Arrays:
Allow the arrays to equilibrate to room temperature (10 to 15 min).
2. Unwrap the arrays and place on the bench top.
3. For each array:
1.
A. Based on the sample size, mark the front side of the array 1A to 24A (for AT channel) and 1C to
24C (for GC channel) only if all 24 samples are being hybridized. Each sample uses two arrays so
take care in labeling the array clearly to ensure that you know which sample is loaded onto each
array.
B. Open the Batch Registration Excel File and scan the Array barcodes onto the “Barcode” column
of the Batch Registration File. Save the Batch Registration File and close it.
Chapter 6 | OncoScan® FFPE Assay Kit Protocol
72
C. Flip the arrays face down and label the array in the same manner as described in Step 3A above
on the back side of the array as well.
D. Insert a 200 μL pipette tip (unfiltered tips are preferred) into the upper right septum.
IMPORTANT: To ensure that the data collected during scanning is associated with the
correct sample, mark the pairs of arrays by sample number (1A through 24A or 1C through
24C).
Figure 6.23 Preparing the Arrays for Hybridization
Prepare the Hybridization Master Mix
To Prepare the Hybridization Master Mix:
NOTE: Prepare only enough hybridization cocktail for the arrays that are being hybridized.
Hyb plate must be prepared fresh right before hybridization. If the samples are not going to
be hybridized on the arrays, store them as HaeIII digest at –20°C.
The number of arrays to be hybridized will depend on the availability of the GeneChip®
Hybridization Oven 645, the number of Fluidics Stations (FS450) for the Wash/Stain procedure
and the Scanner. Please plan ahead for array hybridizations.

The Negative Control sample that was processed until the HaeIII digest will not be
hybridized on the arrays. Hence the total number of arrays hybridized per run will be two
less than the respective Post-GapFill reactions.
Table 6.30 Hybridization Master Mix
Number of Runs per Assay Kit
4
3
2
1
Number of Samples (includes 1 positive control per run)
6
8
12
24
Negative Control per Run
1
1
1
1
x 2 Channels
x 2 Channels
x 2 Channels
x 2 Channels
AT & GC Channels
Reagent
P/N
For 1 Array
12 Arrays
16 Arrays
24 Arrays
48 Arrays
Water
902253
30.0 μL
0.504 mL
0.648 mL
0.936 mL
1.80 mL
Hybridization Mix
902264
118.0 μL
1.9824 mL
2.5488 mL
3.6816 mL
7.08 mL
148.0 μL
2.4864 mL
3.1968 mL
4.6176 mL
8.88 mL
Total
1.
Label a 15 mL conical tube as Hyb and place on ice until ready to use.
Chapter 6 | OncoScan® FFPE Assay Kit Protocol
73
Using the appropriate column in Table 6.30, make the hybridization cocktail by adding volume of
water and hybridization mix to the Hyb tube.
3. Vortex at maximum speed for 3 seconds (1-2 seconds at a time), spin down briefly, and place it on
ice until ready to be used.
2.
Prepare the Hyb Plate and Denature the Samples
To Prepare the Hyb Plate and Denature the Samples:
Remove the HaeIII Plate from the thermal cycler after the program is complete. Ensure the seal is
secure. Vortex at maximum speed for 5 sec. Spin down the plate at 2400 rpm for 60 sec, and place in
a cold block on ice.
2. Label a fresh 96-well plate with Hyb and following the same layout as the 1st PCR Plate, label row A (or
A & E if running 24 samples) as “AT” with a Blue marker and Row C (or C & G if running 24 samples)
as “GC” with a Red marker. Mark the reaction wells in the same colors. Place on ice covered loosely with
a seal.
1.
NOTE: Depending on the volume, if necessary use a reagent reservoir to aliquot the
hybridization cocktail. For smaller volumes, aliquot the hybridization cocktail directly into
the hybridization plate.
NOTE: The negative control is assessed only through the HaeIII gel and will not be carried
through to the Hyb Plate.
If using a reservoir, place a reagent reservoir on ice, and pour the Hybridization Master Mix slowly
and carefully into the reservoir.
4. Using a 12-channel P200 pipette:
3.
A. Aliquot 148 μL of Hybridization Master Mix to the appropriate wells of the Hyb Plate
(Figure 6.24).
IMPORTANT: Any time fresh pipette tips are used, aspirate and dispense three times
to wet the tips prior to aliquoting Hybridization Master Mix.
Chapter 6 | OncoScan® FFPE Assay Kit Protocol
74
Figure 6.24 Adding Hybridization Master Mix to Plate
7 Samples
A
1
2
3
4
5
+
6
7
8
13 Samples
9
10
11
Hyb
12
AT
B
A
1
2
3
4
GC
7
8
9
10
11
Hyb
12
+ AT
+ GC
C
D
D
E
E
F
F
G
G
H
H
9 Samples
1
2
3
4
5
6
7
+
8
25 Samples
9
10
11
Hyb
12
AT
B
C
6
B
+
C
A
5
A
1
2
3
4
5
6
7
8
9
10
11
Hyb
12
AT
B
+
GC
C
D
D
E
E
F
F
G
G
H
H
GC
+ AT
+ GC
Ensure the HaeIII Plate and the Hyb Plate are at the correct orientation (Well A1 at top left). Remove
the seal from the HaeIII Plate carefully.
6. Using a 12-channel P200 pipette, transfer 22 μL of each reaction from the HaeIII Plate to the Hyb
Plate. Pipet up and down 3 times to rinse tips (Figure 6.25).
5.
NOTE: Total volume in each well: 170 μL.
Chapter 6 | OncoScan® FFPE Assay Kit Protocol
Figure 6.25 Transfer from HaeIII Plate to Hyb Plate
7 Samples
A
1
2
3
4
+ _
5
6
B
7
13 Samples
8
9
10
11
A
B
+ _
C
HaeIII
12
AT
GC
C
D
D
E
E
F
F
G
G
H
H
1
2
3
4
1
2
3
4
5
+
6
7
8
9
10
11
Hyb
12
AT
9
10
11
HaeIII
12
+ AT
+ GC
_
1
A
+
GC
2
3
4
5
6
7
8
9
10
11
Hyb
12
+ AT
+ GC
C
D
D
E
E
F
F
G
G
H
H
9 Samples
1
2
3
4
5
6
7
B
25 Samples
+ _
8
9
10
11
HaeIII
12
AT
1
A
2
3
GC
D
E
E
F
F
G
G
H
H
3
4
5
6
7
8
9
10
11
HaeIII
12
AT
GC
+ AT
_
+ GC
_
22 μL
2
5
C
D
1
4
B
+ _
C
22 μL
6
7
+
8
9
10
11
Hyb
12
AT
A
1
2
3
4
5
6
7
8
9
10
11
Hyb
12
AT
B
B
C
8
B
C
A
7
22 μL
B
A
6
_
22 μL
A
5
+
GC
C
D
D
E
E
F
F
G
G
H
H
GC
+ AT
+ GC
75
Chapter 6 | OncoScan® FFPE Assay Kit Protocol
76
Tightly seal the Hyb Plate, vortex at maximum speed for 5 sec, then spin down at 2400 rpm for 60 sec.
8. Place the Hyb Plate on the thermal cycler and make sure the plate is at the correct orientation (well
A1 at top left).
9. Start the OncoScan Hybridization program and allow the program to run.
7.
Figure 6.26 OncoScan Hybridization Thermal Cycler Program
95°C
10 min
49°C
49°C
5 min
∞
Generate and Upload a Sample Batch Registration File (if it was done earlier)
To Generate and Upload a Sample Batch Registration File:
1.
If using Affymetrix® GeneChip® Command Console® (AGCC) software generate and enter
information into a sample batch registration file.
NOTE: Sample nomenclature is extremely important when using the “auto-generate
results file name” function in OncoScan® Console. Please refer to the OncoScan® Console
User Guide (P/N 703195) for recommended sample nomenclature.
A. Scan the array barcodes.
B. Upload the sample and array information to AGCC. For more information, see Generating a
Sample Batch Registration File on page 93.
2. If using a Dx2 system, enter each individual sample as described in the AMDS software manual on
page 33.
Chapter 6 | OncoScan® FFPE Assay Kit Protocol
77
Load the Samples onto OncoScan® Arrays
To Load the Samples Onto Arrays:
At the end of the 5 min incubation at 49°C while leaving the plate on the thermal cycler at 49°C, use
a razor blade to carefully cut the adhesive seal to expose 8 wells at a time (1A through 4A and 1C
through 4C).
2. Working with 8 arrays at a time:
1.
A. Aspirate 160 μL from one well and slowly inject it into the lower left septum of the
correspondingly marked array.
B. Remove the pipette tip from the upper right septum of each array.
C. Repeat Step A and Step B until 8 arrays are loaded.
D. Cover both septa with large Tough-Spots as shown in Figure 6.27.
Using larger Tough-Spots that overlap the window makes them easier to remove later on.
Figure 6.27 Covering the septa with Tough-Spots® that overlap the window
E. Place the arrays into hybridization oven trays.
F. Load the trays into the hybridization oven.
G. Repeat these steps until all of the samples are loaded onto arrays and placed in the oven.
Allow the arrays to incubate at 49°C and 60 rpm for 16 to 18 hours.
4. Discard the HaeIII Plate ONLY if all of the samples were used for hybridization. If there are samples
remaining on the plate, seal the plate well and store it at –20°C.
5. Discard the Hyb Plate in a hazardous waste bin.
3.
IMPORTANT: Arrays must rotate in the oven for 16 to 18 hours at 49°C and 60 rpm (18 hr
maximum before you begin Chapter 7, Washing, Staining and Scanning Arrays).
Chapter 7
Washing, Staining and Scanning Arrays
This chapter describes how to wash, stain and scan the Affymetrix® OncoScan® Arrays. The instruments
that you will use include the:
 GeneChip® Fluidics Station 450 to wash and stain the arrays
 GeneChip® Scanner 3000 7G or GeneChip® Scanner 3000 Dx2 to scan the arrays
Once the arrays are scanned, the array image (.dat file) is ready for analysis.
NOTE: Affymetrix recommends a weekly cleaning protocol (Bleach_450) for the fluidics
station using sodium hypochlorite bleach. Failing to run the Bleach protocol may result in
reduced signal on the OncoScan® Arrays. Refer to Chapter 8, Fluidics Station Care and
Maintenance on page 87 for instructions on maintaining the Fluidics Stations.
Equipment and Consumables Required
The following equipment and consumables are required for washing, staining and scanning Affymetrix®
OncoScan® Arrays.
Table 7.1 Equipment and Consumables Required for Washing, Staining and Scanning Arrays
Item
Vendor
Part Number
GeneChip® Scanner 3000 7G or 3000 Dx2
Affymetrix
—
GeneChip® Fluidics Station 450 or 450Dx
Affymetrix
—
The instrument control application: Affymetrix GeneChip®
Command Console v.3.2.2 or higher or AMDS v.1.3 and higher
Affymetrix
—
Tube, Safe-Lock Tube 1.5 m, Amber
Eppendorf
022363221
Tube, Safe-Lock Tube 1.5 mL, Blue
Eppendorf
022363247
Tube, Safe-Lock Tube 1.5 mL, Natural
Eppendorf
022363352
Rainin Pipetman®
(or equivalent)
—
—
—
Cole-Parmer
H-06418-04
USA Scientific
9185-0000
Pipets, (P-2, P-20, P-200, P-1000)
Sterile-barrier pipette tips and non-barrier pipette tips
Tygon ® Tubing, 0.04” inner diameter
®
Tough-Spots , Label Dots (3/8”)
Chapter 7 | Washing, Staining and Scanning Arrays
79
Reagents Required
The following reagents are required for washing and staining arrays. These reagents have been tested and
evaluated by Affymetrix scientists.
Table 7.2 Reagents Required for Washing and Staining Arrays
Reagent
Stain Buffer 1
Stain Buffer 2
Affymetrix® GeneChip® Array Holding Buffer
Affymetrix® GeneChip® Wash A
Affymetrix® GeneChip® Wash B
Fluidics Station and Scanner Control Software
If you are using the Affymetrix GeneChip Command Console software (version 3.2.2 or higher) to
operate the fluidics and scanner, please refer to the Affymetrix® GeneChip® Command Console® User Guide
(P/N 702569).
If you are using the Affymetrix Molecular Diagnostic Software (version 1.3 or higher) for washing,
staining and scanning the OncoScan arrays, please refer to the AMDS User Guide.
Prime the Fluidics Station
Priming ensures the lines of the fluidics station are filled with the appropriate buffers and the Fluidics
Station is ready to run fluidics protocols.
Priming should be done:
 When the fluidics station is turned on to wash the arrays
 When wash solutions are changed
 Before washing, if a shutdown has been performed
 If the LCD window instructs the user to prime
The Fluidics Station 450 or 450 Dx is used to wash and stain the OncoScan® Arrays.
To Prime the Fluidics Station:
1.
2.
3.
4.
5.
Turn on the Fluidics Station.
Put the wash buffer lines in the appropriate wash buffers (Wash-A line in Wash Buffer-A and WashB line in Wash Buffer-B.
Ensure there is a minimum of 400 mL of Wash-A and 300 mL of Wash-B in the bottles before
starting the “Prime” protocol.
Ensure the water line is in the Deionized Water bottle and there is at least 300 mL of water in the
bottle and that the waste container has been emptied.
Prime the Fluidics Station.
 From Affymetrix Command Console application, start the ‘Affymetrix Launcher’ on the desktop
(Figure 7.1).
 From the Affymetrix Launcher, open ‘AGCC Fluidics Control’ application (Figure 7.2).
Chapter 7 | Washing, Staining and Scanning Arrays

80
From the AGCC Fluidics Control panel, select PRIME_450 script for the specific fluidics stations
and the modules.
IMPORTANT: Use the Affymetrix® GeneChip® Wash A and Wash B buffers that are
designated for the OncoScan® Assay only. These wash buffers differ from the GeneChip®
Expression buffers.
Figure 7.1 Launching the AGCC Fluidics Station Control Software
Affymetrix Launcher desktop icon
Launcher window
Figure 7.2 Affymetrix GeneChip® Command Console® Fluidics Control window
To initiate the fluidics script, click the “Run” icon for each module or click the “Run All” icon, for all
the selected stations and modules. (“Run All” can be performed only after selecting the stations and
modules, pressing the “Copy to Selected Modules” button to copy the protocol to all of the modules.)
7. Once the “prime” protocol is done, lift the needle lever and remove the vials used for priming.
6.
Chapter 7 | Washing, Staining and Scanning Arrays
81
Washing and Staining Arrays
Briefly vortex the stain bottles before aliquoting the reagents.
2. Aliquot the following reagents into 1.5 mL microfuge tubes for each array:
1.
A. Aliquot 500 μL Stain Buffer 1 into 1.5 mL microfuge tubes (use amber color tubes as Stain Buffer
3.
4.
5.
6.
7.
1 is light sensitive).
B. Aliquot 500 μL Stain Buffer 2 into 1.5 mL microfuge tubes (clear/natural tubes).
C. Aliquot 1000 μL Array Holding Buffer into 1.5 mL microfuge tubes (blue tubes).
Select the “OncoScan” wash protocol from the AGCC Fluidics Control Panel.
Start the protocol and follow the instructions displayed on the LCD panel on the Fluidics Station
module.
If you are unfamiliar with inserting and removing arrays from the fluidics station modules, refer to
the appropriate Fluidics Station User’s Guide or Quick Reference Card (P/N 08-0093 for the Fluidics
Station 450).
Eject the wash block to avoid sensor time out.
Remove any previously loaded empty vials.
When prompted to “Load vials 1-2-3”:
A. Place one vial containing 500 μL Stain Buffer 1 in position 1.
B. Place one vial containing 500 μL Stain Buffer 2 in position 2.
C. Place one vial containing 1000 μL Array Holding Buffer in position 3 (Figure 7.3).
Figure 7.3 Reagent positions on the Fluidics Station
Stain 1
in position 1
Array Holding Buffer
in position 3
Stain 2
in position 2
8.
After 16 to 18 hrs of hybridization, remove only the number of arrays that will be washed at this time.
Leave the remaining arrays in the hyb oven at 49°C until the Fluidics Station(s) is ready for another
run. Remove the Tough-Spots from the arrays that are ready to be washed and stained.
IMPORTANT: Tough-Spots must be removed from arrays prior to washing and staining.
Failure to do so can cause inadequate seating of the array in the washblock and failure of
the washing and staining process.
IMPORTANT: Once the arrays are removed from the hybridization oven, quickly load them
onto the Fluidics Station. Delays during this step will impact data quality.
9.
Immediately insert the arrays into the designated modules of the fluidics station while the cartridge
lever is in the Down or Eject position. Pull the handle to engage the array in the washblock, as
directed on the LCD panel.
Chapter 7 | Washing, Staining and Scanning Arrays
82
10. When the LCD panel instructs, press down on the needle lever to snap needles into position and to
start the run.
The fluidics protocol begins. The Fluidics Station dialog box at the workstation terminal and the
LCD window display the status of the washing and staining steps.
NOTE: Watch the fluidics for 2 minutes to make sure the protocol begins. If the user
engages the washblock or presses the needles down before instructed on the LCD panel,
the protocol may not begin. The user should watch for each module to start the process
before walking away from the instrument.
It is strongly recommended to check on the Fluidics Stations every 30 minutes to make
sure there are no errors while washing or staining.
11. When the wash and stain procedure is completed, remove the arrays from the fluidics station by
pressing down the cartridge lever to the Eject position.
12. Check the array window for bubbles or air pockets. If air bubbles are present, return the array to the
fluidics station. Follow the instructions on the LCD panel of the fluidics station. Pull the lever up
and load to remove bubbles.
13. If air bubbles are still present then repeat the above process or use the manual process.
A. Insert a 200 μL pipette tip into the upper right septum of the array.
B. Using a P-200 pipette, remove the entire volume of the holding buffer from the array.
C. Manually fill the array with 170 μL of Array Holding Buffer.
14. If the array has no bubble, it is ready for scanning. Proceed to Scanning Arrays on page 82.
If the arrays cannot be scanned promptly, store them at 4°C in the dark until ready for scanning. Scan
must be performed within 24 hours.
15. Pull up on the cartridge lever to engage wash block. Remove the microcentrifuge vials containing
stain and replace with three empty vials as prompted.
16. If there are more arrays to wash and stain, repeat Step 3 through Step 15 above.
17. When all array washing and staining is complete, shut down the fluidics station following the
procedure on page 85.
Scanning Arrays
The GeneChip Scanner 3000 7G is controlled by AGCC software. The GeneChip Scanner 3000 Dx2 is
controlled by AMDS.
Prepare the Scanner
Turn on the scanner at least 10 minutes before use.
WARNING: The scanner uses a laser and is equipped with a safety interlock system. Defeating
the interlock system may result in exposure to hazardous laser light.
Read and be familiar with the operation of the scanner before attempting to scan an array.
Refer to the GeneChip® Scanner 3000 Quick Reference Card (P/N 08-0075).
Chapter 7 | Washing, Staining and Scanning Arrays
83
Prepare Arrays for Scanning
To Prepare Arrays for Scanning:
If the arrays were stored at 4°C, allow them to warm to room temperature before scanning.
2. If necessary, clean the glass surface of the array with a non-abrasive towel or tissue before scanning.
Do not use alcohol to clean the glass surface.
3. On the back of the array cartridge, clean excess fluid from around the septa, if present.
4. Carefully cover both septa with Tough-Spots (Figure 7.4).
Press to ensure the spots remain flat. If the spots do not apply smoothly (e.g. if you see bumps,
bubbles, tears or curled edges) do not attempt to smooth out the spot. Remove the spot and apply a
new spot.
1.
NOTE: We recommend using the smaller 3/8” Laser Tough-Spots for this step. When
applying the Tough-Spots do NOT overlap the center area as the Tough-Spots should be
flat against the array to avoid being pulled off inside the scanner.
Figure 7.4 Applying Tough-Spots® to Arrays
1A
1C
Chapter 7 | Washing, Staining and Scanning Arrays
84
Scanning the Array
To Scan Arrays:
Ensure the completed OncoScan Batch Registration (Excel file with array barcodes) has been uploaded
to the scanner computer.
Open the ‘AGCC Scan Control’ application from the ‘Affymetrix Launcher’.
2. Load the arrays into the Autoloader of the scanner.
3. Once all the arrays are loaded, click the “Start” icon to initiate the scan.
4. Select the check box “arrays in carousel positions 1-4 at room temperature” (Figure 7.5). If the arrays
are not at room temperature, do not select this option. The scanner will wait 10 minutes before
scanning begins to allow the arrays to reach room temperature.
1.
Figure 7.5 GeneChip Scanner Message
Only one scan per array is required. Pixel resolution and wavelength are preset and cannot be
changed. Once the scan starts, the scanning status is shown in the Scan Status column (Figure 7.6).
Each array takes approximately 7 minutes to scan.
WARNING: The door is locked while the instrument is scanning. Do not attempt to open
the door manually.
Figure 7.6 Scan Status
Chapter 7 | Washing, Staining and Scanning Arrays
85
Adding Arrays During an Autoloader Run
To Add Arrays While an AutoLoader Run is in Progress:
1.
Click the Add Chips icon
.
The GeneChip Scanner message appears.
Figure 7.7 GeneChip Scanner Message
2.
Click Add after Scan.
IMPORTANT: Do not use the Add Now feature. Use only the Add after Scan feature when
working with OncoScan® Arrays.
When the status on the scanner reads Autoloader Door Unlocked, open the scanner and add the
arrays.
4. Close the scanner.
5. When the following message is displayed, click OK.
3.
Figure 7.8 GeneChip Scanner Message
After you click OK, click the Resume icon.
7. If any arrays in the carousel are to be rescanned, select the check box Allow rescans.
6.
NOTE: If you select Allow rescans, all arrays in the carousel will be scanned. Remove
arrays that have already been scanned and do not need to be rescanned. If you do not
select Allow rescans and you leave already scanned arrays in the scanner, you will see
an error message in the scanner status stating the array has already been scanned and has
been skipped. This is normal.
Chapter 7 | Washing, Staining and Scanning Arrays
86
Shutting Down the Fluidics Station
To Shut Down the Fluidics Station:
1.
2.
3.
4.
5.
6.
7.
Gently lift up the cartridge lever to engage (close) the washblock.
After removing an array from the holder, the LCD window displays the message ENGAGE
WASHBLOCK. The instrument automatically performs a cleanout procedure. The LCD window
indicates the progress of this procedure.
When REMOVE VIALS is displayed on the LCD display window, remove the vials.
The REMOVE VIALS message indicates the cleanout procedure is complete.
If no other processing is to be performed, place the wash lines into a bottle filled with deionized
water.
Using AGCC, choose the Shutdown_450 protocol for all modules.
Run the protocol for all modules.
The Shutdown protocol is critical to instrument reliability. Refer to the instrument User’s Guide for
more information.
When the protocol is complete, turn the instrument off. Close the lid on the Wash Buffer bottles and
save them at room temperature for later use.
Empty the waste bottle.
IMPORTANT: To maintain the cleanliness of the fluidics station and obtain the highest quality
image and data possible, a weekly bleach protocol is highly recommended (see Chapter 8,
Fluidics Station Care and Maintenance on page 87.
Chapter 8
Fluidics Station Care and Maintenance
General Fluidics Station Care






Use a surge protector on the power line to the fluidics station.
Always run a Shutdown protocol when the instrument is unused overnight or longer. This will prevent
salt crystals from forming within the fluidics system.
To ensure proper functioning of the instrument, perform periodic maintenance.
When not using the instrument, leave the sample needles in the lowered position. Each needle should
extend into an empty vial. This will protect them from accidental damage.
Always use deionized water to prevent contamination of the lines. Change buffers with freshly prepared
buffer at each system startup.
The fluidics station should be positioned on a sturdy, level bench away from extremes in temperature
and away from moving air.
WARNING: Before performing any maintenance, turn off power to the fluidics station to
avoid injury in case of a pump or electrical malfunction.
Fluidics Station Bleach Protocol
Affymetrix recommends a weekly cleaning protocol for the fluidics station. This protocol uses commonly
purchased sodium hypochlorite bleach.
This protocol is designed to eliminate any residual SAPE-antibody complex that may be present in the
fluidics station tubing and needles. The protocol runs a bleach solution through the system followed by
a rinse cycle with deionized (DI) water. This protocol takes approximately one hour and forty minutes to
complete. Affymetrix recommends running this protocol weekly, regardless of the frequency of use. The
current version of the protocol can be found at:
www.affymetrix.com/support/technical/fluidics_scripts.affx
The Bleach Cycle
To avoid carryover, or cross contamination, from the bleach protocol, Affymetrix recommends the use of
dedicated bottles for bleach and DI water. Additional bottles can be obtained from Affymetrix.
Table 8.1 Affymetrix Recommended Bottles
Part Number
Description
400118
Media Bottle, SQ, 500 mL
400119
Media Bottle, SQ, 1000 mL
Disengage the washblock for each module by pressing down on the cartridge lever. Remove any probe
array cartridge Figure 8.1 on page 88.
2. Prepare 500 mL of 0.525% sodium hypochlorite solution using deionized water.
You can follow these directions to make 500 mL of bleach:
In a 1 liter plastic or glass graduated cylinder, combine 43.75 mL of commercial bleach (such as
Clorox® bleach, which is 6.15% sodium hypochlorite) with 456.25 mL of DI H2O, mix well. Pour the
solution into a 500 mL plastic bottle, and place the plastic bottle on fluidics station.
1.
Chapter 8 | Fluidics Station Care and Maintenance
88
IMPORTANT:

The shelf life of this solution is 24 hr. After this period, you must prepare fresh solution.

Each fluidics station with 4 modules requires 500 mL of 0.525% sodium hypochlorite
solution.
Figure 8.1 Disengaged Washblocks Showing Cartridge Levers in the Down
Position. Remove any cartridges.
3.
As shown in Figure 8.2 on page 89:
A. Place on the fluidics station an empty one liter waste bottle, a 500 mL bottle of bleach and a one
liter bottle of DI water.
The Bleach protocol requires approximately one liter of DI water.
B. Insert the waste line into the waste bottle.
C. Immerse all three wash and water lines into the bleach solution.
IMPORTANT: Do NOT immerse the waste line into the bleach.
Open the instrument control software (AGCC or AMDS).
5. Choose the current bleach protocol for each module.
4.
Chapter 8 | Fluidics Station Care and Maintenance
89
Figure 8.2 Bleach Cycle
Immerse the tubes into
the 0.525% sodium
hypochlorite solution.
The waste line remains
in the waste bottle.
Figure 8.3 Fluidics Station Protocol Window: Select All Modules
6.
In AGCC, run the protocol for all modules.
NOTE: The fluidics station will not start until the needle lever is pressed down (Figure 8.4 on
page 90). The temperature will ramp up to 50°C.
Follow the prompts on each LCD display window. Load empty 1.5 mL vials onto each module if not
already done so.
8. Press down on each of the needle levers to start the bleach protocol (Figure 8.4).
7.
Chapter 8 | Fluidics Station Care and Maintenance
90
Figure 8.4 Press Down on the Needle Levers to Start the Bleach Protocol
The fluidics station will begin the protocol, emptying the lines and performing the cleaning cycles
using bleach solution.
10. After approximately 30 minutes, the LCD display window will prompt you when the bleach cycle is
over and the rinse cycle is about to begin.
9.
The Rinse Cycle
Once the bleach cycle has finished, the second part of the protocol is a rinse step. This step is essential
to remove all traces of bleach from the system. Failure to complete this step can result in damaged arrays.
Follow the prompts on the LCD display window for each module. Lift up on the needle levers and
remove the bleach vials. Load clean, empty vials onto each module.
2. Remove the three wash and water lines from the bleach bottle and transfer them to the DI water
bottle (Figure 8.5).
At this step, there is no need to be concerned about the bleach remaining in the lines.
1.
Chapter 8 | Fluidics Station Care and Maintenance
91
Figure 8.5 Immerse the Three Wash and Water Lines in the DI Water Bottle
Press down on the needle levers to begin the rinse cycle.
The fluidics station will empty the lines and rinse the needles.
4. When the rinse is completed after approximately one hour, the fluidics station will bring the
temperature back to 25°C and drain the lines with air.
The LCD display will read CLEANING DONE.
5. Discard the vials used for the bleach protocol.
6. After completing the bleach protocol, follow the suggestions for storage of the Fluidics Station 450
in Table 8.2.
3.
Chapter 8 | Fluidics Station Care and Maintenance
Table 8.2 Storage Suggestions for the Fluidics Station 450
If:
Then do this:
Planning to use the
system immediately
After running the bleach protocol, remove the DI water supply used in the rinse phase
and install the appropriate reagents for use in the next staining and washing protocol
(including fresh DI water).
 Perform a prime protocol without loading your probe arrays.
Failure to run a prime protocol will result in irreparable damage to the loaded
hybridized probe arrays.
Not planning to use the
system immediately
Since the system is already well purged with water, there is no need to run an
additional shutdown protocol.
Remove the old DI water bottle and replace it with a fresh bottle.
Not planning to use the
system for an extended
period of time (longer
than one week)
Remove the DI water and perform a “dry” protocol shutdown. This will remove most
of the water from the system and prevent unwanted microbial growth in the supply
lines.
Also, remove the pump tubing from the peristaltic pump rollers.
92
Appendix A
Registering Samples in Affymetrix® GeneChip® Command Console®
IMPORTANT: We strongly recommend that you batch register your sample and array
information prior to washing and scanning. If you accidentally wash and scan your arrays
without first registering them, the sample file (.ARR files) will not include two of the
attributes required by OncoScan® Console: sample type and consented markers. Before you
can genotype the .CEL files, you will have to manually edit these files to include the required
information.
Generating a Sample Batch Registration File
To Generate a Sample Batch Registration File:
From the Launcher, open AGCC Portal.
2. Hold the cursor over Samples tab and select Batch Register from the drop-down menu (Figure A.1).
1.
Figure A.1 Selecting Batch Register to open a spreadsheet template.
3.
Create a blank Batch Registration File by selecting (Figure A.2 on page 94):
A. A template (this example uses the template included with OncoScan Console).
B. The file type (TSV or Excel).
C. The number of samples to be recorded on the spreadsheet.
D. Optional: A project name from the drop-down menu.
E. Optional: The array type from the drop-down menu (select OncoScan).
Appendix A | Registering Samples in Affymetrix® GeneChip® Command Console®
94
Figure A.2 Creating a blank batch sample registration file.
4.
Click Download.
A blank registration file is displayed.
IMPORTANT: Sample nomenclature is very important for downstream use of Auto-Generate
OSCHP file names in OncoScan Console. Common root names should be consistent all the way
up to the last character of the cel file name. Affymetrix recommends using an “A” or “C” as
the last character to designate the channel in the CEL file naming convention. Example:
“SampleName_05A.CEL” is an AT Channel file, while “SampleName_05C.CEL” is a GC Channel
file.
Enter the sample information (Figure A.3 on page 94).
Required fields:
 Sample File
 Project
 Sample File Name (name that AGCC will assign to the .ARR file)
 Array Name
 Probe Array (click in an empty cell to open the drop-down menu)
 Barcode
6. Open File  Save As and:
5.
A. Select the location where the file will be stored.
B. Enter a name for the file.
Figure A.3 Enter information into the batch registration file.
Appendix A | Registering Samples in Affymetrix® GeneChip® Command Console®
95
Upload the Batch Registration File to AGCC
To Upload the Batch Registration File to AGCC:
Open the batch registration file created for this set of samples.
Scan the array barcodes into this file.
3. In the Batch Sample Registration window, Step 3 (Figure A.4 on page 95):
1.
2.
A. If custom barcodes have been affixed to the arrays, select the check box Allow Custom Barcodes.
B. Click Browse; then navigate to and open the batch registration file.
C. Click Upload.
D. When you see the message “Do you want to save samples?” click Save (Figure A.5 on page 96).
Figure A.6 on page 96 appears.
IMPORTANT: You must click Save once you have uploaded the batch registration file.
If you do not click Save, the information is not uploaded.
Figure A.4 Uploading a sample batch registration file.
2
3
Appendix A | Registering Samples in Affymetrix® GeneChip® Command Console®
Figure A.5 Batch registration.
Figure A.6 Batch registration
96
Appendix A | Registering Samples in Affymetrix® GeneChip® Command Console®
97
About Batch Registration Files
A batch registration file is a file that contains sample attributes, such as the sample name, source plate,
source well and sample type. The options for creating this file include:
 Using the pre-defined template provided with OncoScan Console (OncoScan.TEMPLATE).
 Editing a template.
 Creating your own template.
Using the Predefined Template — OncoScan.TEMPLATE
If the predefined template is not already installed on the computer with AGCC, you must first copy it to
this computer. Obtain the OncoScan Console software from Affymetrix. The .ZIP file containing the
installer also includes the file OncoScan.TEMPLATE. Copy this file to the Templates folder used by
AGCC (usually located at C:\Command_Console\Templates).
Creating or Editing a Template
Editing Templates
To Edit a Template:
From the AGCC Portal, open Administration  Templates  Edit.
2. Select the template you would like to edit (for example, you can edit OncoScan.Template to suit your
project or study requirements).
3. Delete, edit, or add attributes.
4. Click Save.
1.
Appendix A | Registering Samples in Affymetrix® GeneChip® Command Console®
98
Creating Templates
You can create and edit templates in AGCC Portal. To help with template creation, the following fields
are automatically included when creating a template:
Path
Probe Array Type
Barcode
Project
Sample File Name
Array Name
To Create Your Own Template:
1.
From the AGCC Portal, open Administration  Templates  New.
Figure A.7 Creating a new template for sample batch registration
2.
Enter a name for the template; then click Next.
The template name is appended to each attribute that you add; therefore, you may want to keep the
name as short as possible (see Figure A.11 on page 100).
Figure A.8 Entering a name for the new template
3.
Add attributes to the template by clicking Add and defining the attribute.
Appendix A | Registering Samples in Affymetrix® GeneChip® Command Console®
99
Figure A.9 Adding attributes to a new template
4.
When finished adding attributes, click Save.
Figure A.10 Adding attributes to a new template
The template as described in the instructions above would look like Figure A.11 when selected for use as
an Excel spreadsheet.
Appendix A | Registering Samples in Affymetrix® GeneChip® Command Console®
Figure A.11 Example of a custom template
100
Appendix B
Thermal Cycler Programs
Seven thermal cycling programs are used throughout the OncoScan® FFPE Assay Kit Protocol. This
appendix describes each of the programs required for the protocol.
Thermal Cyclers
To run the OncoScan® FFPE Assay Kit Protocol at a throughput of 24 assays/day, you will need at least
2 thermal cyclers: 1 in the Pre-Amp Lab, and 1 in the Post-Amp Lab.
Pre-PCR Lab Thermal Cycler Programs
Set up the thermal cyclers in the Pre-PCR Lab to run the following programs:
 OncoScan Anneal
 OncoScan Gap Fill (This program is set up differently in the Veriti Thermal Cycler)
 OncoScan 1st PCR
Post-PCR Lab Thermal Cycler Programs
Set up the thermal cyclers in the Post-PCR Lab to run the following programs:
 OncoScan 2nd PCR
 OncoScan HaeIII
 OncoScan Hybridization
Appendix B | Thermal Cycler Programs
102
Setting the Ramp Speed and Volume for Each Program
IMPORTANT: Set the correct ramp speed and volume for each thermal cycler program.
Ramp Speed
Max = Ramp speed for GeneAmp® PCR System 9700 Thermal Cycler (gold or silver block). Use the
instructions below for setting up the “9700-Max-Mode” ramp speed on the Veriti Thermal Cycler.
Ramp Speed Settings for Veriti® Thermal Cycler
Use the “Convert Method” feature of the Veriti Thermal Cycler to create a method for the Veriti Thermal
Cycler with ramp rates that simulate those on the 9700 thermal cycler at Max mode. Please follow
instructions on the Veriti® Thermal Cycler User Guide, Chapter 5 (pages 5-2 to 5-4).
To Use a Run Method From a GeneAmp® PCR System 9700 Thermal Cycler
1.
2.
3.
4.
5.
6.
7.
8.
Obtain a copy of the original method you want to convert, either by printing it from the instrument
or copying it manually to a piece of paper.
In the Main Menu screen, touch Tools Menu.
In the Tools Menu screen, touch Convert a Method to open the Convert Method wizard.
Touch the check box next to the run method’s original format “9700 Max Mode” then touch Next.
Veriti® Thermal Cycler User Guide (page 5-3).
In the second page, enter the original run method. Touch Next when you are done.
In the Save Run Method page, enter a name and select a folder for the run method. Names are limited
to 16 characters. By default, the name begins with “9700-Max-Mode-”.
Also enter the reaction volume, the temperature for the heated cover as 105.0°C, and any notes for
this method.
When you finish, touch Save & Exit. The Tools Menu is displayed.
Important Information on Using the Veriti® Thermal Cycler
The touch screen sometimes may not respond when “Pause Run” or “Resume Run” functions are
performed. Make sure the screen is only touched ONCE, firmly, and NOT TAPPED TWICE.
When the touch screen is touched with a double tap to perform “Pause Run”, the program will
momentarily pause and then immediately resume. This can create assay interruptions and possibly lead
to assay failures. Ensure the touch screen is only touched ONCE, firmly.
Pay attention to the screen to make sure:
 the program pauses when “Pause Run” is performed. (Figure B.1)
 the program resumes when “Resume Run” is touched. (Figure B.1)
 When the program is Paused, the clock STOPS on “Time Remaining”, displayed in the upper right
corner of the touch screen. (Figure B.1)
Appendix B | Thermal Cycler Programs
103
Figure B.1 Veriti® Thermal Cycler Touch Screen
When the program is Paused,
the clock STOPS on “Time
Remaining” display.
The program pauses when
“Pause Run” is performed.
The program resumes when
“Resume Run” is touched.
Make sure to:
 Set the Default Standby Time-Out to 00.00 (screen does not get turned off)
 Set the Default Pause Duration to 24:00:00 (pause time: 24 hours)
Refer to the Figure B.2, below, for detailed instructions on setting time-outs.
Figure B.2 Setting Time-Outs for the Veriti® Thermal Cycler
Setting Time-Outs
1. In the Main Menu screen, touch Settings Menu.
2. In the Settings Menu screen, touch Set Time-Outs.
3. In the Set Time-Outs screen, touch Time-out, then
enter the duration.
 Default Standby Time-Out sets the duration that
the instrument is idle before the touchscreen is
turned off. Enter a duration of 00:00 if you do not
want the instrument to turn off the touchscreen.
 Default Pause Duration sets the duration the
instrument pauses, after you touch Pause during a
run. Set to 24:00:00 (pause time: 24 hours).
4. Touch Done, then OK to save your changes and
return to the Settings Menu screen.
Appendix B | Thermal Cycler Programs
104
OncoScan® Anneal Thermal Cycler Program
About the OncoScan® Anneal Program
The OncoScan Anneal program consists of three holds and no cycles.
Ramp speed and volume:
 Ramp speed
 GeneAmp PCR System 9700 (gold or silver block) = Max
 Volume: 10 μL
IMPORTANT: The ramp speed and volume must be set the first time you use the program.
See Setting the Ramp Speed and Volume for Each Program on page 102.
Table B.1 Stages of the OncoScan Anneal Thermal Cycler Program
Stage
Temperature
Time
Denature
95°C
5 min
Anneal
58°C
16 - 18 hrs
Anneal
58°C
Infinity
Figure B.3 OncoScan Anneal Thermal Cycler Program
95°C
5 min
58°C
16 - 18 hr
Pause the cycler,
chill for 1 min and
Spin plates
58°C
∞
Appendix B | Thermal Cycler Programs
105
OncoScan® Gap Fill Thermal Cycler Program
About the OncoScan® Gap Fill Program
The OncoScan Gap Fill thermal cycler program consists of 10 holds and 1 cycle.
 Ramp speed
 GeneAmp PCR System 9700 (gold or silver block) = Max
 Volume: 42 μL
IMPORTANT: The ramp speed and volume must be set the first time you use the program.
See Setting the Ramp Speed and Volume for Each Program on page 102.
Table B.2 Stages of the OncoScan Gap Fill Thermal Cycler Program
Stage
Temperature
Time
Cycles
Gap Fill
58°C
11 min
—
AT/GC Mix Addition
58°C
11 min
—
Exo Mix Addition
37°C
20 min
Denature
95°C
10 min
Cleavage Mix Addition
37°C
15 min
Denature
95°C
15 min
Hold
4°C
Infinity
Figure B.4 OncoScan Gap Fill Thermal Cycler Program (for GeneAmp® PCR System 9700)
Pause the cycler at
the dotted lines
Remove the plate at the end
of 58°C and then allow the
cycler to ramp down to 37°C
95°C
95°C
15 min
10 min
58°C
58°C
11 min 11 min
37°C
37°C
20 min
15 min
4°C
∞
+4 μL
+ 3 μL
dNTP mix (A/T) Exo mix
or
(well 17.0 μL)
+ 25 μL
Cleavage Mix
(well 42.0 μL)
dNTP mix (G/C)
(well 14.0 μL)
Blue arrows indicate reagent addition steps
Appendix B | Thermal Cycler Programs
106
NOTE: For the Veriti Thermal Cycler, when programming the “OncoScan Gap Fill” program,
combine the first two 58°C incubations for 11 min each into one stage at 58°C for 22 min (see
Table B.2 and Figure B.5). This change is recommended because it has been noticed that the
touch screen does not consistently respond to the “PAUSE RUN” function when paused
between the two stages of 58°C. (The touch screen may not pause at the end of the 58°C at
11 min and will move on to the next 58°C stage). This will ruin the Gap Fill reaction, leading
to assay failure. Using a laboratory timer is critical during the first two 58°C incubations. Make
sure the program pauses when the “PAUSE RUN” is touched.
Figure B.5 OncoScan Gap Fill Thermal Cycler Program (for Veriti Thermal Cycler)
Pause the cycler at
the dotted lines to add the
reagents for the next step
95°C
Remove the plate at the end
of 58°C and then allow the
cycler to ramp down to 37°C
95°C
15 min
10 min
58°C
22 min
37°C
37°C
20 min
15 min
4°C
∞
+4 μL
+ 3 μL
dNTP mix (A/T) Exo mix
or
(well 17.0 μL)
+ 25 μL
Cleavage Mix
(well 42.0 μL)
dNTP mix (G/C)
(well 14.0 μL)
Blue arrows indicate reagent addition steps
Appendix B | Thermal Cycler Programs
107
OncoScan® 1st PCR Thermal Cycler Program
About the OncoScan® 1st PCR Program
The OncoScan 1st PCR program consist of 2 holds and 1 cycle.
Ramp speeds:
 GeneAmp PCR System 9700 (gold or silver block) = Max
Volume: 67 μL
IMPORTANT: The ramp speed and volume must be set the first time you use the program.
See Setting the Ramp Speed and Volume for Each Program on page 102.
Table B.3 Stages of the OncoScan 1st PCR Thermal Cycler Program
Stage
Temperature
Time
PCR Reaction Start
60°C
30 sec
Template Denaturation
95°C
1 min
Denaturation
95°C
20 sec
Anneal
60°C
10 sec
Extension
72°C
10 sec
Extension
72°C
5 min
Hold
4°C
Infinity
Figure B.6 OncoScan 1st PCR Thermal Cycler Program
95°C
95°C
1 min 20s
72°C
60°C
60°C
30s
10s
10s
72°C
5 min
20 cycles
4°C
∞
Cycles
20 cycles
Appendix B | Thermal Cycler Programs
108
OncoScan® 2nd PCR Thermal Cycler Program
About the OncoScan® 2nd PCR Program
The OncoScan 2nd PCR program consist of 2 holds and 1 cycle.
Ramp speeds:
 GeneAmp PCR System 9700 (gold or silver block) = Max
Volume: 27 μL
IMPORTANT: The ramp speed and volume must be set the first time you use the program.
See Setting the Ramp Speed and Volume for Each Program on page 102.
Table B.4 Stages of the OncoScan 2nd PCR Thermal Cycler Program
Stage
Temperature
Time
PCR Reaction Start
60°C
30 sec
Template Denaturation
95°C
1 min
Denaturation
95°C
20 sec
Anneal
60°C
10 sec
Extension
72°C
10 sec
Extension
72°C
5 min
Hold
4°C
Infinity
Figure B.7 OncoScan 2nd PCR Thermal Cycler Program
95°C
95°C
1 min 20s
72°C
60°C
60°C
30s
10s
10s
72°C
5 min
15 cycles
4°C
∞
Cycles
15 cycles
Appendix B | Thermal Cycler Programs
109
OncoScan® HaeIII Digest Thermal Cycler Program
About the OncoScan® HaeIII Digest Program
The OncoScan HaeIII Digest program consists of 2 holds and no cycles.
Ramp speeds:
 GeneAmp PCR System 9700 (gold or silver block) = Max
Volume: 30 μL
IMPORTANT: The ramp speed and volume must be set the first time you use the program.
See Setting the Ramp Speed and Volume for Each Program on page 102.
Table B.5 Stages of the HaeIII Digest Thermal Cycler Program
Stage
Temperature
Time
HaeIII Digestion
37°C
88 min
Continuation of HaeIII Digestion after
pausing to get the QC gel aliquot
37°C
2 min
Deactivation of the Enzyme
95°C
10 min
Hold
4°C
Infinity
Figure B.8 HaeIII Digest Thermal Cycler Program
95°C
10 min
37°C
37°C
88 min
2 min
4°C
Pause at 88 min to
remove QC gel aliquot
∞
Appendix B | Thermal Cycler Programs
110
OncoScan® Hybridization Thermal Cycler Program
About the OncoScan® Hybridization Program
The OncoScan Hybridization program consists of 2 holds and no cycles.
Ramp speeds:
 GeneAmp PCR System 9700 (gold or silver block) = Max
Volume: 170 μL
IMPORTANT: The ramp speed and volume must be set the first time you use the program.
See Setting the Ramp Speed and Volume for Each Program on page 102.
Table B.6 Stages of the OncoScan Hybridization Thermal Cycler Program
Stage
Temperature
Time
Denature
95°C
10 min
49°C
5 min
49°C
Infinity
Standby
Figure B.9 OncoScan Hybridization Thermal Cycler Program
95°C
10 min
49°C
49°C
5 min
∞
Appendix C
Equipment, Software, Consumables and Reagents List
This chapter lists the equipment, software, consumables and reagents required to perform the
OncoScan® FFPE Assay Protocol.
Required from Affymetrix®
Affymetrix Equipment and Software Required
Table C.1 Affymetrix Equipment and Software Required

Item
Part Number

GeneChip® Fluidics Station 450 — two or more units required
00-0079

Tubing, Silicone peristaltic for GeneChip® Fluidics Station 450
Contact Affymetrix

GeneChip® Hybridization Oven 645
00-0331

GeneChip® 3000 Scanner 7G System with Workstation and Autoloader
00-0215

GeneChip® Command Console® Software (AGCC)
Version 3.2 or higher

OncoScan® Console (OC) Software analysis (AGCC)
Latest version
Appendix C | Equipment, Software, Consumables and Reagents List
OncoScan® Reagents and OncoScan® Arrays Required
Table C.2 OncoScan® Reagent Kit and OncoScan® Arrays

Item

OncoScan® FFPE Assay Kit Array and Reagent Kit Bundle, Sufficient for 24 samples and
includes 48 OncoScan Arrays — Components in this kit are listed below.
Part Number
OncoScan® Somatic Mutation Probe Mix 1.0

Somatic Mutation Probe Mix 1.0
OncoScan® Copy Number Probe Mix 1.0 & Controls


Positive Control (12ng/μL)
Negative Control


Copy Number Probe Mix 1.0
Buffer A
OncoScan® Gap Fill and 1st Stage PCR






Buffer A
Gap Fill Enzyme Mix
SAP, Recombinant (1 U/μL)
dNTP Mix (A/T)
dNTP Mix (G/C)
Nuclease-free Water





Exo Mix
Cleavage Buffer
Cleavage Enzyme (2 U/μL)
PCR Mix
Taq Polymerase (5 U/μL)
OncoScan® 2nd Stage PCR and Post PCR Processing




PCR Mix
Taq Polymerase (5 U/μL)
Buffer B
HaeIII Enzyme (10 U/μL)

Exo I Enzyme (20 U/μL)
Nuclease-free Water
Hybridization Mix

Array Holding Buffer

Wash B


OncoScan® Stain Reagents


Stain 1
Stain 2
Individual Bottles


Wash A
Arrays

OncoScan® Array, 4 x 12 pack
902293
112
Appendix C | Equipment, Software, Consumables and Reagents List
113
From Other Suppliers — Reagents, Equipment and Consumables Required
Reagents Required for Gel QC
(Not included in the OncoScan® FFPE Assay Kit)
Table C.3 Reagents Required for Gel QC (Not included in the OncoScan FFPE Assay Kit)
Item
Supplier
Part Number
Bio-Rad
161-3062
50 bp DNA ladder
New England Biolabs
N3236S
Gel loading Dye, Blue (6X)
New England Biolabs
B7021S
100% Glycerol
Affymetrix
16374
EDTA, 0.5 M Solution
Affymetrix
15694
1X TE buffer Low EDTA, pH 8.0
Affymetrix
75793
3% 96-well pre-cast agarose gel
(as needed, based on the number of samples)
Table C.4 Reagents and Equipment Required for Gel QC using E-Gels (Not included in the OncoScan FFPE Assay Kit)
Item
Supplier
Part Number
Mother E-Base™ Device
Life Technologies
EB-M03
Daughter E-Base™ Device
(optional for running multiple gels simultaneously)
Life Technologies
EB-D03
E-Gel® 48 4% Agarose Gels
Life Technologies
G8008-04
TrackIt™ Cyan/Orange Loading Buffer
Life Technologies
10482-028
New England Biolabs
N3236S
Affymetrix
71786
50 bp DNA ladder
Nuclease-free Water
Appendix C | Equipment, Software, Consumables and Reagents List
114
Miscellaneous Consumables Required
Table C.5 Miscellaneous Consumables Required
Item
Qty Needed per
24 Samples
Part Number
Supplier
10
1402-2400
USA Scientific
1/2” Laser Tough-Spots
as needed
RNBW-1100
Diversified Biotech
3/8” Laser Tough-Spots
as needed
RNBW-1000
Diversified Biotech
Micro-Amp Clear Adhesive Film
as needed
4306311
Applied Biosystems
Optical 96 Well Half-skirt plate
5
289196
E&K Scientific
MicroAmp Optical 96 well reaction plate (Optional for
UK; compatible with ABI thermal cyclers)
5
N8010560
Invitrogen
Optional
(for gel QC)
82006-636
VWR
1.5 mL Safe-Lock Tubes, Natural
as needed
022363204
Eppendorf
1.5 mL Safe-Lock Tubes, Blue
as needed
022363247
Eppendorf
1.5 mL Safe-Lock Tubes, Amber
as needed
022363221
Eppendorf
Non-Stick RNase-free Microfuge Tubes (1.5 mL)
as needed
AM12450
Life Technologies
Non-Stick RNase-free Microfuge Tubes (2.0 mL)
as needed
AM12475
Life Technologies
25 mL Reagent Reservoir
as needed
89094-662
VWR
Pipette Tip Refills, 20 μL
as needed
GP-L10F
Rainin
Pipette Tip Refills, 200 μL
as needed
GP-L200F
Rainin
Pipette Tip Refills, 1000 μL
as needed
GP-L1000F
Rainin
21008-103
VWR
0.2 mL PCR 12-tube strip with 12-cap strips, natural
VWR PCR Plate, 96-Well, Flat Plate
15 mL Centrifuge tubes
Appendix C | Equipment, Software, Consumables and Reagents List
115
Equipment Required
When performing the pre-PCR stages of the OncoScan FFPE Assay Kit Protocol, great care should be
taken to avoid sample contamination with PCR products.
Table C.6 Equipment Required
Item
Part Number
Supplier
Optional Part Number/
Manufacturer
Needed In
Needed In
Vortexer Maxi Mix II
M37615
Thermo Scientific
Vortex Genie G560 /VWR
Pre-PCR Lab
Post-PCR Lab
Picofuge Galaxy Mini
C1213
VWR
—
Pre-PCR Lab
Post-PCR Lab
Striptube Centrifuge Galaxy Mini
C1213
VWR
—
Pre-PCR Lab
Post-PCR Lab
89231-998
VWR
—
Pre-PCR Lab
Post-PCR Lab
—
Pre-PCR Lab
Post-PCR Lab
Ice Pan, Maxi
96-well Cold Blocks
GeneAmp PCR system 9700
Thermal Cycler (with Gold block)
81001
N050200
Life Technologies
4375786* (Veriti 96-well)
/ Life Technologies
Pre-PCR Lab
Post-PCR Lab
Single Channel Pipettes
(entire LTS set)
L-2, L-20,
L-200, L-1000
Rainin
—
Pre-PCR Lab
Post-PCR Lab
12-Channel LTS Pipette
(20 μL and 200 μL)
L12-20,
L12-200
Rainin
—
Pre-PCR Lab
Post-PCR Lab
24-Channel LTS Pipette
(20 μL and 200 μL)
L24-20,
L24-200
Rainin
—
Pre-PCR Lab
Post-PCR Lab
MicroAmp Clear Adhesive Film
4306311
Life Technologies
—
Pre-PCR Lab
Post-PCR Lab
Plate Centrifuge 5810 or 5804
5810 / 5804
Eppendorf
—
Pre-PCR Lab
Post-PCR Lab
Benchtop Cooler
89004-558
VWR
—
Pre-PCR Lab
Post-PCR Lab
Gel Box (Wide-Mini sub cell GT
basic system)
170-4489
Bio-Rad
—
—
Post-PCR Lab
Power Supply for Gel
Electrophoresis
164-5070
Bio-Rad
—
—
Post-PCR Lab
Varies (Inquire)
Protein Simple
—
—
Post-PCR Lab
00-0331
Affymetrix
—
—
Post-PCR Lab
Probe Array Cartridge Carriers
90-0356 to
90-0359
Affymetrix
—
—
Post-PCR Lab
GeneChip® Fluidics Station 450
Varies
depending on
location
Affymetrix
—
—
Post-PCR Lab
GeneChip® Scanner 3000 7G
System with Workstation and
AutoLoader
Varies
depending on
location
Affymetrix
—
—
Post-PCR Lab
Inquire
Affymetrix
—
—
Post-PCR Lab
AlphaImager HP System for gel
imaging (with software)
Hybridization Oven 645
GeneChip® Command Console®
Software (AGCC)
* The Veriti 96-well Fast Thermal Cycler, P/N 4375305 is not compatible with this assay protocol. Use only one of the two recommended
thermal cyclers listed above.
Appendix C | Equipment, Software, Consumables and Reagents List
116
Table C.6 Equipment Required (Continued)
Item
Part Number
Supplier
Optional Part Number/
Manufacturer
Needed In
Needed In
If preparing samples using QIAGEN FFPE Kit
Fume Hood
—
Thermomixer® R with 1.5 mL
adapter
5355-31595
Eppendorf
—
Centrifuge 5415D
5425 50003
Eppendorf
5415C
Vortexer Maxi Mix II
M37615
Thermo Scientific
Picofuge Galaxy Mini
C1213
VWR
DNA120-115
Thermo Scientific
DNA110
Fluoroskan Ascent Microplate
Fluorometer
5210470
Thermo Scientific
—
Single Channel Pipettes (entire
LTS set)
L-2, L-20,
L-200, L-1000
Rainin
—
DNA120 Speedvac
Vortex Genie G560 /VWR Sample Preparation Lab (Clean
or Pre-PCR)
—
Supplier Contact List
Table C.7
Supplier Contact List
Supplier
Web Site Address (www not required for some addresses)
Affymetrix
www.affymetrix.com
Applied Biosystems
www.appliedbiosystems.com
Bio-Rad
www.bio-rad.com
Bio-Smith
www.biosmith.com
C.B.S. Scientific
www.cbsscientific.com
Clontech
www.clontech.com
Diversified Biotech
divbio.com
E&K Scientific
www.eandkscientific.com
Eppendorf
www.eppendorf.com
ESCO
www.escoglobal.com
Fisher Scientific
www.fishersci.com
Invitrogen
www.lifetechnologies.com
Lonza
www.lonza.com
Life Technologies
www.lifetechnologies.com
New England Biolabs
www.neb.com
QIAGEN
www.qiagen.com
Rainin
www.rainin.com
TekNova
www.teknova.com
VWR
www.vwr.com
Appendix D
Running QC Gels on E-Gel
Procedure for Running First (1st PCR) and Second (HaeIII) QC Gel on E-Gel
The instructions given below are guidelines to run E-gels to check the quality of the PCR and HaeIII
digest products that are generated in the OncoScan® FFPE Assay. For additional information, please
refer to the E-Gel® Pre-Cast Agarose Electrophoresis System.
http://tools.lifetechnologies.com/content/sfs/productnotes/F_071215_E-Gel-RD-MKT-TL-HL.pdf
Equipment, E-Gels, and Reagents Required
Table D.1 Equipment, E-Gels, and Reagents Required
Item
Supplier
Part Number
Mother E-Base™ Device
Life Technologies
EB-M03
Daughter E-Base™ Device
(optional for running multiple gels simultaneously)
Life Technologies
EB-D03
E-Gel® 48 4% Agarose Gels
Life Technologies
G8008-04
TrackIt™ Cyan/Orange Loading Buffer
Life Technologies
10482-028
New England Biolabs
N3236S
Affymetrix
71786
50 bp DNA ladder
Nuclease-free Water
CAUTION: The E-Gel® contains Ethidium Bromide. Always wear gloves when handling E-Gels
and dispose of the gel and gloves in a hazardous waste container.
Prepare and Run the First QC Gel (1st PCR)
CAUTION: When transferring sample volumes from one 96-well plate to another 96-well
plate, make sure BOTH plates are in the correct orientation (well A1 at top left) before
pipetting.
Prepare the Gel QC Reagents
Prepare 1:1000 dilution of TrackIt™ Cyan/Orange Loading Buffer by adding 50 μL of TrackIt Cyan/
Orange Loading Buffer to 49.5 mL of Nuclease-free water.
2. Prepare a working stock of 1:10 dilution of NEB 50 bp DNA ladder as given below.
1.
Table D.2 Preparation of 1:10 Dilution of NEB 50 bp DNA Ladder (Working Stock)
Reagent
Volume
Water
85.0 μL
TrackIt Cyan/Orange Loading Buffer (Undiluted)
5.0 μL
NEB 50 bp DNA ladder (1000 ng/ μL)
10.0 μL
Total Volume
100.0 μL
Appendix D | Running QC Gels on E-Gel
118
The final concentration of the ladder is 100 ng/μL. The volume prepared above (100 μL) is adequate
for loading ~30 lanes. Store at 2-8°C and use it within one month.
3. Prepare the diluted 50 bp DNA ladder enough to load 20 μL per lane.
 Volume per lane: Add 3 μL of diluted ladder prepared above (Step 2) to17 μL of water.
 If less than 25 samples were run on the assay, prepare 80 μL of the diluted ladder. (The ladder will
be loaded on both sides of a 48-well gel in the Marker lane.)
 If 25 samples were run on the assay, prepare 40 μL of the diluted ladder. (It is recommended to
load the diluted ladder in the Marker lane on left side of a 48-well gel only, so the Marker lane on
the right side of the gel can be used to run negative control.)
Prepare the Gel Plate
1.
Label a new PCR plate Gel1 (the 1st QC Gel Plate) and load it as follows:
A. Add 16 μL of 1:1000 diluted TrackIt Cyan/Orange Loading Buffer in the appropriate wells
B. Add 4 μL of 1st PCR product in the wells of the Gel1 Plate that contains loading buffer.
Pipet up and down three times to rinse the tips.
2. Seal 1st PCR Plate tightly and keep in a cold block on ice.
3. Tightly seal the first gel QC plate, vortex and spin down.
Run the E-Gel
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
Turn on the power for the E-Base (red light).
Push the Power/Prg button to make sure the program is set to EG mode (not EP).
Remove the comb(s) from a 48-well 4% Agarose E-Gel and wipe away any buffer that comes out of
the gel or is on the surface.
Insert the E-Gel into the slot.
Load 18 μL of the 1st PCR sample onto the 48-well 4% agarose E-Gel.
Load 18 μL of the diluted marker prepared above (Refer to Step 3 in Prepare the Gel QC Reagents
section) into each of the marker wells on either both sides or one side of the gel as needed.
Fill all empty wells with 20 μL of water.
Ensure program is set to EG mode and Set run time to 20 min.
Push the Power/Prg button again to start (light will change from red to green).
When the run time is reached, the system will automatically shut off (the dye should be near the end
of the lane). The gel is now ready for imaging.
Examine the 1st PCR QC gel in a gel imager to ensure one single band of the PCR product at
approximately 120 bp is observed.
Figure D.1 illustrates good QC gel results.
Appendix D | Running QC Gels on E-Gel
119
Figure D.1 For samples in which successful amplification has occurred, one single band at approximately120 base
pairs should be seen. No distinct band at approximately 120 base pairs should be visible in the Negative Control and
in samples in which amplification has not occurred. The image below was taken with a 1.0 second exposure setting.
Prepare and Run the Second QC Gel (HaeIII Digest)
CAUTION: When transferring sample volumes from one 96-well plate to another 96-well
plate, make sure BOTH plates are in the correct orientation (well A1 at top left) before
pipetting.
Prepare the Gel QC Reagents
Use the 1:1000 dilution of TrackIt Cyan/Orange Loading Buffer prepared in Step 1 of Prepare the
Gel QC Reagents on page 117.
2. Use the 1:10 dilution of NEB 50 bp DNA ladder prepared in Step 2 of Prepare the Gel QC Reagents
on page 117.
3. Prepare the diluted 50 bp DNA ladder enough to load 20 μL per lane.
 Volume per lane: Add 3 μL of diluted ladder prepared above (Step 2) to17 μL of water.
 If less than 25 samples were run on the assay, prepare 80 μL of the diluted ladder. (The ladder will
be loaded on both sides of a 48-well gel in the Marker lane.)
 If 25 samples were run on the assay, prepare 40 μL of the diluted ladder. (It is recommended to
load the diluted ladder in the Marker lane on left side of a 48-well gel only, so the Marker lane on
the right side of the gel can be used to run negative control.)
1.
Appendix D | Running QC Gels on E-Gel
120
Prepare the Second QC Gel (HaeIII Digest) Plate
1.
2.
3.
4.
5.
6.
7.
8.
9.
Label a new PCR plate Gel2 (the HaeIII QC Gel Plate) and load it as follows:
Add 16 μL of 1:1000 diluted TrackIt Cyan/Orange Loading Buffer to the appropriate wells.
When the cycler reaches 88 min at 37°C, pause the cycler.
Remove the HaeIII Plate and place it on a cold block for 1 minute.
Ensure the HaeIII Plate is tightly sealed and vortex at max speed for 5 seconds. Spin down the plate
at 2400 rpm for 60 seconds.
Ensure the plate is at the correct orientation (well A1 at top left). Remove the plate seal slowly and
carefully.
Remove 4 μL of HaeIII digest sample and add it to the Gel2 Plate into the appropriate wells of the
Gel1 Plate containing the loading buffer. Pipet up and down three times to rinse the tips.
Seal the HaeIII Plate and place it back on the thermal cycler. Resume the OncoScan HaeIII program.
Tightly seal the first gel QC plate, vortex and spin down.
Run the E-Gel
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
Turn on the power for the E-Base (red light).
Push the Power/Prg button to make sure the program is set to EG mode (not EP).
Remove the comb(s) from a 48-well 4% Agarose E-Gel and wipe away any buffer that comes out of
the gel or is on the surface.
Insert the E-Gel into the slot.
Load 18 μL of the HaeIII digest sample onto the 48-well 4% Agarose E-Gel.
Load 18 μL of the diluted marker prepared above (Refer to Step 3 in Prepare the Gel QC Reagents
section) into each of the marker wells on either both sides or one side of the gel as needed.
Fill all empty wells with 20 μL of water.
Ensure program is set to EG mode and Set run time to 20 min.
Push the Power/Prg button again to start (light will change from red to green).
When the run time is reached, the system will automatically shut off (the dye should be near the end
of the lane). The gel is now ready for imaging.
Examine the HaeIII QC gel in a gel imager to ensure that a predominant pattern of doublet bands
around 40 bp and 70 bp are observed.
Figure D.2 illustrates good QC gel results.
Appendix D | Running QC Gels on E-Gel
121
Figure D.2 The gel indicates both successful amplification during the second stage PCR reaction and HaeIII digestion.
The predominant pattern should be two bands at approximately 40 and 70 bp. The image below was taken with a 1.0
second exposure setting.
Appendix E
FFPE DNA Extraction Protocol for OncoScan® Assay
NOTE: Affymetrix strongly recommends using the QIAamp DNA FFPE Tissue Kit protocol for
purifying DNA from FFPE Blocks that will be used in OncoScan® Assay. For improved DNA
yields, we also recommend a modification to the QIAamp DNA FFPE Tissue Kit protocol. The
modified procedure adds a heating step at 98°C for 15 minutes to improve the tissue digestion
process to release DNA from tissue sections
Please refer to the QIAamp DNA FFPE Tissue Kit protocol for more information on the QIAGEN web
page given below.
http://www.qiagen.com/Products/Catalog/Sample-Technologies/DNA-Sample-Technologies/
Genomic-DNA/QIAamp-DNA-FFPE-Tissue-Kit#resources
Equipment and Reagents Required, but not Provided with OncoScan® FFPE Assay
Kit
When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles.
For more information, consult the appropriate safety data sheets (SDSs), available from the product
supplier.
Equipment Required
Table E.1 Equipment Required
Equipment
Vendor
P/N
Microtome (HM340E Electronic Rotary Microtome Package, with
Universal Cassette Clamp and Disposable Blade Carrier E (ThermoFisher Cat#:
905190A))
VWR International
89219-568
Vortex Genie 2 (G560)
VWR International
58815-234
Microcentrifuge, 5424 (EPPE022620401)
VWR International
80094-126
Thermomixer R (22670107) (Qty Required: 2)
VWR International
21516-166
Block for 24 x 1.5 mL Tubes for Thermomixer R (22670522) (Qty Required: 2)
VWR International
21516-176
Pipet-Lite LTS Pipette, Single Channel, 0.5-10 μL
Rainin
L-10
Pipet-Lite LTS Pipette, Single Channel, 20-200 μL
Rainin
L-200
Pipet-Lite LTS Pipette, Single Channel, 100-1000 μL
Rainin
L-1000
Pipet-Lite LTS Pipette, 12-Channel, 2-20 μL
Rainin
L12-20
Appendix E | FFPE DNA Extraction Protocol for OncoScan® Assay
123
Consumables Required
Table E.2 Consumables Required
Equipment
Vendor
P/N
Fischer Scientific
3153735
Eppendorf tubes, 1.5 mL microcentrifuge tubes
VWR International
022363204
Non-Stick RNase-free Microfuge Tubes (1.5 mL)
Life Technologies
AM12450
Greenpak LTS 20 μL Filter Tip, 960 Tips
Rainin
GP-L10F
Greenpak LTS 200 μL Filter Tip, 960 Tips
Rainin
GP-L200F
Greenpak LTS 1000 μL Filter Tip, 768 Tips
Rainin
GP-L200F
VWR PCR Plate, 96-Well, Flat Plate
VWR
82006-636
HP-35 High-Profile blades with PTFE coating
Reagents Required
Table E.3 Reagents Required
Equipment
Vendor
QIAamp DNA FFPE Tissue Kit
 QIAamp MinElute Columns
 Collection Tubes - 2 mL
 Buffer ATL
 Proteinase K
 Buffer AL
 Buffer AW1
 Buffer AW2
 Buffer ATE
QIAGEN
P/N








56404
1020901
1016810
1014758
19133
1014604
1014790
1014592
1049476
Xylene
Sigma Aldrich
534056-500ML
Ethanol (96-100%)
Sigma Aldrich
459836
QIAGEN
1007885
RNase A
Optional Prerequisite
The yield of tissue from FFPE is highly dependent on the type of tissue and method used for the initial
fixation. For optimal DNA yield, samples submitted for extraction should have a tissue size of 400-700
square mm.
Preparation of Buffers
Preparing Buffer ATL
 Check whether precipitate has formed in Buffer ATL. If necessary, dissolve by heating to 70°C with
gentle agitation.
Preparing Buffer AL
 Check whether precipitate has formed in Buffer AL. If necessary, dissolve by heating to 70°C with
gentle agitation.
Appendix E | FFPE DNA Extraction Protocol for OncoScan® Assay
124
Preparing Buffer AW1
 Add 25 mL ethanol (96-100%) to the bottle containing 19 mL Buffer AW1 concentrate. Can be stored
in room temperature up to 1 year.
Preparing Buffer AW2
Add 30 mL ethanol (96-100%) to the bottle containing 13 mL Buffer AW2 concentrate. Can be stored
in room temperature up to 1 year.

Deparaffinization
1.
From an FFPE block, prepare 10 micron slices. Place 5 slices in a 1.5 mL Eppendorf Safe-Lock Tube.
NOTE: The Eppendorf Tubes must have the centrifugation stability of up to 30,000 x g to
prevent tube breakage. Please refer to the consumables list.
2.
3.
4.
5.
6.
Turn on two thermal mixers. Set one to 56°C and the other to 98°C.
Add 1 mL of Xylene to the tube.
Vortex the tube at max speed for 10 seconds.
Spin down the tube at full speed (~14000 rpm) for 5 minutes.
Without disturbing the pellet remove the Xylene.
NOTE: Be sure to place waste Xylene in the appropriate container.
Add 1 mL of Ethanol to the tube.
8. Vortex the tube at max speed for 10 seconds.
9. Spin down the tube at full speed (~14000 rpm) for 5 minutes.
10. Without disturbing the pellet remove the Ethanol by using a P1000 pipette.
7.
NOTE: Be sure to place waste Ethanol in the appropriate container.
11. Repeat Step 7 through Step 10 once more.
12. Spin down the tube at full speed (~14000 rpm) for 3 minutes.
13. Use a p20 or P200 pipette to completely remove any residual Ethanol without disturbing the pellet.
14. Allow any remaining Ethanol to evaporate by letting the tube air dry for 10 minutes at room
temperature.
Tissue Lysis
Add 180 μL of ATL Buffer to the tube after ensuring the residual ethanol has completely evaporated.
Vortex at full speed for 10 seconds.
3. Spin down the tube briefly and then place it onto the thermal mixer that is set at 98°C.
4. Incubate the tube for 15 minutes with a 15 second mix at 1400 rpm every 1 minute.
1.
2.
NOTE: Thermomixer settings: Ensure the thermomixer is programmed to shake the
samples for 15 sec after each minute at 1400 RPM.
After 15 minutes, stop the thermomixer program and turn off the thermomixer. Let the tube cool
down for 5 minutes in the thermomixer before removing them.
This is to ensure the vials do not pop open due to high heat and pressure.
6. Remove the tube carefully and slowly (as they may still pop open) from the thermal mixer and allow
it cool at room temperature for 10 minutes.
7. Spin down the tube to remove any solution from the top of the tube.
5.
Appendix E | FFPE DNA Extraction Protocol for OncoScan® Assay
8.
9.
10.
11.
12.
125
Add 20 μL of Proteinase K to the tube.
Vortex the tube at max speed for 10 seconds, then spin down briefly.
Place tube on the thermal mixer that is set at 56°C.
Incubate the tube for at least 3.5 hours with a 15 second mix at 1400 rpm every 1 minute.
After 3.5 hours, verify that all tissue has lysed.
A. If tissue remains, incubate the samples overnight. If tissue still remains, add an additional 20 μL
of Proteinase K and continue incubation for a minimum of 1 hour.
13. Spin down the tube and place them onto the thermal mixer that is set at 90°C.
14. Incubate the tube for 1 hour with a 15 second mix at 1400 rpm every 1 minute.
NOTE: Thermomixer settings: Ensure the thermomixer is programmed to shake the
samples for 15 sec after each minute at 1400 RPM.
15. After 1 hour, remove the tube from the thermal mixer and allow to cool at room temperature for
10 minutes.
16. Spin down the tube to remove any solution from the top of the tube.
17. Add 2 μL of RNase A to each tube.
18. Vortex the tube at max speed for 10 seconds, then spin down briefly. Allow to incubate for 2 minutes.
DNA Purification
Remove a QIAamp MinElute column from the refrigerator and allow to warm to room temperature
for 15 minutes.
2. Remove the ATL tube and equilibrate at room temperature.
3. Add 200 μL of Buffer AL to the sample tube, vortex at max speed for 10 seconds, then spin down
briefly.
1.
A. If processing multiple samples, ensure that after adding Buffer AL, Ethanol is added as quickly as
possible.
B. Precipitate may form at this step, which does not affect the DNA yield.
4. Add 200 μL of Ethanol to each tube, vortex at max speed for 10 seconds, then spin down briefly.
5. Label the QIAamp MinElute column (in a 2 mL collection tube) appropriately.
6. Carefully transfer the entire lysate to the QIAamp MinElute column (in a 2 mL collection tube)
without wetting the rim, close the lid, and centrifuge at 6000 x g (8000 rpm) for 1 min.
A. Check to see that all the lysate has moved through the column. If lysate is still in the column
centrifuge again at a higher speed for 1 minute.
7. Place the column into new collection tube and discard eluate.
8. Open the column and add 500 μL of Buffer AW1.
9. Load the column onto the centrifuge and spin at 8000 rpm (6000g) for 1 minute.
A. Check to see that the entire Buffer AW1 has moved through the column. If Buffer AW1 is still
in the column centrifuge again at a higher speed for 1 minute.
10. Place the column into new collection tube and discard eluate.
11. Open the column, and add 500 μL of Buffer AW2.
12. Load the column onto the centrifuge and spin at 8000 rpm (6000g) for 1 minute.
A. Check to see that the entire Buffer AW2 has moved through the column. If Buffer AW2 is still
in the column centrifuge again at a higher speed for 1 minute.
13. Place the column into new collection tube and discard eluate.
14. Load the column onto the centrifuge and spin at 14000 rpm (20000g) for 3 minutes to dry the
membrane completely.
Appendix E | FFPE DNA Extraction Protocol for OncoScan® Assay
126
DNA Elution
1.
2.
3.
4.
5.
Label a clean Nuclease-free 1.5 mL tube for DNA elution.
Place the column into the labeled 1.5 mL tube prepared for elution.
Add 50 μL of Buffer ATE to the center of the column membrane.
Close the lid and incubate the column at room temperature for 5 minutes.
Load columns onto the centrifuge and spin at 14000 rpm (20000g) for 1 minute to elute the DNA.
A. Note: Caps for 1.5 mL tube will not be able to close at this step due to the columns. When loading
onto the centrifuge rotate caps to the right to keep them from breaking during the spin.
B. When eluting multiple samples, alternate positions in centrifuge to make room for caps.
Quantitation of Eluted FFPE DNA
Perform either of the two Affymetrix recommended and tested dsDNA quantitation protocols that
are included in the user guide, to measure the concentration of the eluted FFPE DNA. Affymetrix
strongly recommends using these protocols that have been tested at Affymetrix using the following
kits for DNA quantitation.
 Quant-iT ™ PicoGreen® dsDNA Assay Kit (Catalog Number: P7589, LifeTechnologies ™)
 Qubit® dsDNA HS Assay Kit (Catalog Number: Q32851, LifeTechnologies ™)
2. Record the results and the volume information in a spreadsheet.
1.
Appendix F
PicoGreen® dsDNA Quantification Protocol for OncoScan® Samples
Materials Required but not Provided with OncoScan® FFPE Assay Kit
Quant-iT™ PicoGreen® dsDNA Assay Kit: Invitrogen (Life Technologies) P/N P7589
 Equivalent Catalog Number: P/N P11496 (dye product size: 10 x 100 μL)
 Quant-iT™ PicoGreen dsDNA Reagent (200× concentrate, frozen at +4°C in dark): In order to avoid
repeated freeze thawing of the reagent, divide the 1 mL stock solution into 50~100 μL aliquots and use
within one year, or until expiration date. Avoid more than 3 freeze-thaw cycles on any one aliquot vial.
 Lambda DNA Standard Stock (100 ng/μL aqueous at +4°C in screw-cap tube)
Other User Supplied Reagent
 1X TE pH 7.5~8.0 (for blank samples and as diluent for DNA samples): Affymetrix P/N 75893 or
equivalent
Consumables
 P-20 & P-200 pipette tips
 96-well microtiter plates
 384-well dark plate for fluorescence reading: E&K Scientific, P/N EK-30076
 Screw-cap Eppendorf tubes
 8 or 12-length strip tubes
 15 mL conical tubes
 Plate centrifuge
Equipment
 P-20 & P-200 single and multiple-channel pipettes
 Vortexer
 Bench-top mini-centrifuge
 Fluorescence Plate Reader
Genomic DNA Samples
Purified gDNA Samples should be kept refrigerated for no more than 2 weeks, or frozen at –20°C or
lower for long-term storage. If frozen, thaw samples at room temperature for 20-30 minutes; spin down
condensate; vortex 3 seconds; re-spin; vortex an additional 3 seconds; re-spin (it is essential that the DNA
samples are fully resuspended and homogenous. Ensure any DNA that might have dried on the side of
the wells have been collected and completely resuspended.)
Appendix F | PicoGreen® dsDNA Quantification Protocol for OncoScan® Samples
128
Procedure
NOTE: Follow manufacturer's instructions on how to perform the Quant-iT™ PicoGreen®
dsDNA Assay and how to operate the Fluorescence Plate Reader with appropriate software
for data collection and analysis. It is recommended to follow the specific instructions provided
below for accurate and reliable quantification of FFPE-derived DNA samples.
Prepare Lambda DNA Standards
Use the table below to make stock solutions of the lambda DNA standards in screw-cap tubes. Store in
no more than 200 μL aliquots at +4°C as working stock for use within a week, or at –20°C for longerterm. Use 1X TE as diluent. The optimal range of standards may need to be modified for different
fluorescence reading systems.
Table F.1 Picogreen Standards: from Lambda DNA (100 ng/μL in kit)
Standard
Final Conc.
μL Diluent
DNA Used
μL DNA
A
2.0 ng/μL
980
Lambda
20
B
1.5 ng/μL
50
Std A
150
C
1.0 ng/μL
100
Std A
100
D
800 pg/μL
120
Std A
80
E
600 pg/μL
140
Std A
60
F
400 pg/μL
160
Std A
40
G
200 pg/μL
180
Std A
20
H
100 pg/μL
190
Std A
10
Prepare Sample Dilutions
Dilute high concentration DNA samples with 1x TE such that the final concentration is within the
dynamic range of the PicoGreen dsDNA Assay. It is usually acceptable to have diluted DNA
concentrations in the range of 0.3 ~2.0 ng/μL. However, the actual dynamic range can vary significantly
depending on the fluorescence reading system used. It is recommended for each customer to carefully
characterize the appropriate limits of PicoGreen quantification for their specific system.
NOTE: For FFPE-derived DNA samples, it is usually sufficient to start with a 1:50 dilution, or
try multiple dilution levels for each sample. Prepare TWO independent replicate dilution
series of each sample. If a sample is tested at multiple dilution levels, only measured
concentrations within the dynamic range determined above for your instrument will be
accepted.
Mix sample dilutions thoroughly by vortexing for 5 seconds followed by spinning down briefly in a
centrifuge.
Appendix F | PicoGreen® dsDNA Quantification Protocol for OncoScan® Samples
129
Prepare 1:200 Dilution of PicoGreen® Stock in 1X TE
Thaw PicoGreen 200× stock in the dark at room temperature, vortex & spin down.
Determine the total amount of diluted PicoGreen reagent needed.
Mix diluted PicoGreen working solution in Eppendorf or 15 mL conical tube by vortexing. Store the
diluted PicoGreen working solution in dark at room temperature until needed.
Perform the PicoGreen® dsDNA Quantification Assay
Mix each diluted DNA sample with PicoGreen working solution in a 384-well dark plate suitable for
fluorescence reading. Vortex the sealed plate at max speed (2400-3000 rpm) for 5-8 seconds, mixing for
1 second at each corner and then the center. and spin at 2000 rpm for 30 seconds. Incubate the plate in
dark for 5 minutes before reading fluorescence. Be sure to include standards and blanks on the same plate.
Verify the excitation and emission filters of correct wavelengths are selected.
Use appropriate software associated with the fluorescence plate reader for data collection and analysis.
IMPORTANT: When generating the standard curve, it is recommended to use direct linear fit
without logarithmic transformation. If an option is available in the software to specify the
intercept of the linear fit, it is usually beneficial to force the intercept to be zero. Enforce the
quality of the fitted curve by only accepting standard curves with R2 >0.98.
Appendix G
Qubit® dsDNA Quantification Protocol for OncoScan® Samples
Materials Required but not Provided with OncoScan® FFPE Assay Kit
Qubit® dsDNA HS Reagent Kit: Invitrogen (Life Technologies) P/N Q32851 or P/N Q32854
 Qubit® dsDNA HS Reagent (Component A): 250 μL or 1.25 mL 200x concentrate in DMSO (stored
at room temperature in dark)
 Qubit® dsDNA HS Buffer (Component B): 50 mL or 200 mL (stored at room temperature)
 Qubit® dsDNA HS Standard #1 (Component C): 1 mL or 5 mL, 0 ng/μL in TE (stored at +4°C)
 Qubit® dsDNA HS Standard #2 (Component D): 1 mL or 5 mL, 10 ng/μL in TE (stored at +4°C)
Other User-Supplied Reagent
 1X TE pH 7.5~8.0 (as diluent for DNA samples): Affymetrix P/N 75893 or equivalent
Consumables
 P-20 & P-200 pipette tips
 15 mL or 50 mL falcon tube (disposable) for mixing the Qubit® working solution
 0.5 mL Qubit® assay tubes: Invitrogen P/N Q32856 (500 tubes) or Axygen PCR-05-C tubes (VWR,
P/N 10011-830)
Equipment
 Vortexer
 Bench-top mini-centrifuge
 P-20 & P-200 pipettes
 Qubit® 2.0 Fluorometer: Invitrogen (Life Technologies) P/N Q32866
Genomic DNA Samples
Purified gDNA Samples should be kept refrigerated for no more than 2 weeks, or frozen at –20°C for
long-term storage. If frozen, thaw samples at room temperature for 20-30 minutes; spin down
condensate; vortex 3 seconds; re-spin; vortex an additional 3 seconds; re-spin (It is essential that the
DNA samples are fully resuspended and homogeneous. Ensure any DNA that might have dried on the
side of the wells have been collected and completely resuspended.).
Appendix G | Qubit® dsDNA Quantification Protocol for OncoScan® Samples
131
Procedure
NOTE: Follow manufacturer's instructions on how to use the Qubit® dsDNA HS Assay Kit and
the Qubit® 2.0 Fluorometer. It is recommended to follow the specific instructions provided
below for accurate and reliable quantification of FFPE-derived DNA samples.
Sample Pre-dilution
Dilute high concentration DNA samples with 1x TE such that the final concentration is estimated to be
within the range of 0.3 ng/μL ~2.0 ng/μL (equivalent to Qubit measured concentration, or QF value,
between 15 ng/mL and 100 ng/mL).
NOTE: For FFPE-derived DNA samples, it is usually sufficient to start with a 1:50 dilution, or
try multiple dilution levels for each sample to insure that the DNA being measured is within
the 0.3 ng/μL ~2.0 ng/μL range.
Mix sample dilutions thoroughly by vortexing for at least 3 seconds followed by spinning down in a
microcentrifuge.
Use pre-diluted DNA in the Qubit dsDNA HS Assay within one hour at room temperature.
Qubit® dsDNA HS Assay
Follow manufacturer's instructions on how to perform the Qubit® dsDNA HS Assay Kit, with following
notes:
 Although the kit allows a variable sample input volume from 2 to 20 μL, it is recommended to always
use a constant sample volume of 10 μL.
 After DNA has been incubated with the Qubit® working solution for a minimum of 2 minutes, read
fluorescence signal in the Qubit® 2.0 Fluorometer within 30 minutes, or store the assay tube protected
from light for up to 2 hours at room temperature before reading. Do not refrigerate.
 Always generate a new calibration curve using standards processed in parallel with test samples and
with fresh prepared Qubit working solution.
 For accurate quantification, the Qubit measured concentration (QF value) should be between
15 ng/mL and 100 ng/mL (equivalent to sample pre-dilution to 0.3 ng/μL ~2.0 ng/μL, when 10 μL is
used in the assay). If the Qubit measured concentration falls out of the range, adjust the dilution factor
and measure again.
Appendix G | Qubit® dsDNA Quantification Protocol for OncoScan® Samples
132
Normalization of Qubit®-determined FFPE DNA Concentration (Optional)
This additional step is applicable to FFPE-derived DNA samples when it is desired to convert the Qubitdetermined dsDNA concentration to the equivalent concentration determined by the PicoGreen®
dsDNA quantification assay (Invitrogen P/N P11496). Although the two DNA quantification platforms
work by the same mechanism, minor discrepancies may exist in the reported concentration for FFPEderived DNA samples. The PicoGreen® dsDNA quantification assay has been used primarily to
determine the amount of assay input DNA during the development of OncoScan product. Therefore for
best consistency, a normalization factor is recommended to convert the Qubit-determined DNA
concentration to PicoGreen equivalent value.
For Qubit measurements within the recommended dynamic range (QF value between 15 ng/mL and
100 ng/mL; see Sample Pre-dilution above), the following equation can be used:
PicoGreen Equivalent Concentration = Qubit-determined Concentration * 0.8
Example
An FFPE DNA sample is first diluted 50 times with 1X TE. 10 μL of the diluted sample is tested in the
Qubit dsDNA HS assay with 190 μL Qubit working solution (total assay volume 200 μL). The QF value
is 32 ng/mL, without using the “Dilution Calculator” feature of the instrument.
Then (Qubit) Concentration of the diluted sample = 32 * (200/10) = 640 ng/mL
NOTE: This value can also be obtained directly using the “Dilution Calculator” feature.
To convert this value into PicoGreen Equivalent, a normalization factor of 0.8 if applied:
Corresponding PicoGreen Equivalent Concentration = 640 * 0.8 = 512 ng/mL (0.512 ng/μL)
For testing in the OncoScan Assay, the original sample concentration will be stated as:
0.512 * 50 = 25.6 ng/μL
Appendix H
Troubleshooting
General Assay Performance Recommendations
As with any assay requiring PCR, the OncoScan® FFPE Assay Kit protocol has an inherent risk of
contamination from PCR product from the previous reactions. In Chapter 2, Laboratory Setup and
Recommendations (page 10), two separate work areas are strongly recommended to minimize the risk of
cross contamination during the assay procedure. Personnel should not re-enter the Pre-PCR Clean Area
once exposed to PCR products without first showering and changing into clean clothes.
Carefully reading and following the protocol as written is essential. The OncoScan® FFPE Assay Kit
protocol has been validated using the reagents and suppliers listed. Substitution of reagents and/or taking
protocol shortcuts could result in sub-optimal results.
Success of the OncoScan assay depends on the following critical steps recommended in the user guide:
 Accurate sample quantitation by PicoGreen® method
 Proper storage and use of the reagents
 Proper use of the workflow
 Proper use and maintenance of the equipment including
 Calibrated thermal cyclers
 Calibrated Pipettes
 Using filter pipette tips and recommended plate seals
 Maintenance of Fluidics stations – Bleach protocol performed every week and tubing changed every
5-6 weeks
Additional recommendations are as follows:
 Plan ahead to ensure that the reagents and equipment you require, including pipettes, are in the correct
work area. Ensuring the proper equipment is available in the proper laboratory areas will make the
workflow easier, and will help reduce the risk of sample contamination.
 If Veriti Thermal Cyclers are used to run the OncoScan FFPE Assay, pay particular attention to the
details in Appendix B such as converting the programs to 9700-max mode, caution on the touch screen
use while pausing and resuming the program, setting the default Pause time, etc...
 Pay particular attention to the storage and handling of reagents. Proper storage and handling is
particularly important for enzymes such as Gap-fill, Exo Mix and the Taq Polymerase enzyme.
To prevent loss of enzyme activity:
 Store the enzymes in a cooler placed in a –20°C freezer to preserve activity. When taking out enzymes
for reaction setup, always use a cooler chilled to –20°C.
 Take care when pipetting enzymes stored in glycerol, which is viscous. Do not store enzymes at
–80°C.
 Preparing the Master Mixes with 20% to 25% overages as provided in this user guide and the Quick
Reference Card ensures consistency in reagent preparation by minimizing pipetting errors and reducing
handling time of temperature sensitive reagents. The success of this assay depends on the accurate
pipetting and subsequent thorough mixing of small volumes of reagents.
 The PCR reaction for this assay has been validated using the specified thermal cyclers. We highly
recommend that your PCR thermal cyclers be calibrated regularly. Take care when programming your
thermal cycler and ensure all programs in this assay runs at “Max mode” (9700) setting. Use the
recommended 96-well plate and the plate seal. Substitution of thermal cyclers, plates or plate seals are
not recommended as the results could be suboptimal.
Appendix H | Troubleshooting




134
It is essential to run gels to monitor both the First PCR and HaeIII reactions.
For the first PCR reaction, individual PCR products from both AT and GC channels are run on a gel
in adjacent wells. A single band should be visible around 120 bp. See Prepare and Run the First QC Gel
on page 59 for more information.
Following HaeIII digestion, remove the volume required for the gel from each well BEFORE the
cycler reaches 95°C and run the samples on a gel. Successful digestion is confirmed by the presence of
a doublet band at 40 bp and 70 bp. See Prepare and Run the HaeIII Gel (Second QC Gel) on page 68 for
more information.
Always run positive and negative controls in parallel with each group of samples.
The absence of a 120 bp band on the First PCR gel for the negative control confirms no previously
amplified PCR product has contaminated the samples or reagents. Use Genomic DNA from the
OncoScan® Reagent Kit as a positive control in the assay. These controls are effective troubleshooting
tools that will help you confirm the successful completion of each stage of the assay.
Regularly calibrate all single channel and multi-channel pipettes and always use filter pipette tips.
Hybridization oven temperature and the RPM is critical to the performance of the assay. Use the
GeneChip® Hybridization Oven 645 only. Hybridization ovens should be serviced at least once a year
to ensure that they are operating within specification.
OncoScan® FFPE Assay Kit Protocol Troubleshooting
Table H.1 OncoScan® FFPE Assay Kit Protocol Troubleshooting
Problem
Potential Cause
Solution
Low input starting material.
Recheck the quantification for the sample by using the recommended
PicoGreen protocol to make sure you have 80 ng of starting material.
Failed anneal reaction.
Mis-pipetting in the Anneal step by not adding the probe mix or the
input DNA can result in no PCR bands. Repeat Assay from the
beginning.
Sample type.
Check to see if this sample might contain chemical or enzymatic
inhibitors. If so, try cleaning sample over a column and starting assay
from the beginning.
Faint or no PCR product
visible on the 1st PCR QC
gel image in AT or GC
channel for a given
sample.
Pipetting error during Gel QC.
Run the first PCR from both channels again to ensure there was not a
pipetting error in loading the gel.
Pipetting error in Pre-PCR step.
If the repeat gel shows the same faint/no PCR band in a given channel,
there was likely a mis-pipetting step after the Channel Split in the PrePCR stage. Repeat this sample from the beginning of the assay.
Smeared or multiple
bands in PCR Gel
Low input starting material.
Recheck the quantification for the sample by using the recommended
PicoGreen protocol to make sure you have 80 ng of starting material.
PCR product in the
negative control
Reagents or equipment
contaminated with amplified
product.
Always use filter pipette tips. Clean the Pre-PCR Lab and equipment
thoroughly using 10% bleach. Decontaminate the pipettes following
manufacturer’s recommendation. Retrain personnel on pre-lab best
practices. Repeat the assay using fresh reagents and sample. DO NOT
OPEN the seal of the amplified 1st PCR Plate in the Pre-PCR Lab. Do
not store the 1st PCR Plate in the Pre-PCR Lab.
Hae III gel smeared, no
distinct double bands
Forgot to pause the thermal cycler
at 88 min and pull an aliquot for
Gel QC.
Check your gel against the example gel in which the aliquot was taken
after the 95°C incubation.
PCR Gel QC Step
Faint or no PCR product
visible on the 1st PCR QC
gel image in both AT and
GC channel for a given
sample.
Appendix H | Troubleshooting
135
Table H.1 OncoScan® FFPE Assay Kit Protocol Troubleshooting (Continued)
Problem
Potential Cause
Solution
Dim array
Fluidics Stations needs bleach
protocol run.
Dim arrays (low signal on the array) might indicate that the fluidics
stations need the bleach maintenance performed. We recommend
bleaching the fluidics station once a week. The peristaltic tubing needs
to be changed every 5-6 weeks.
The .cel file is not
generated
Signal from corner checkerboards
is dim.
Ensure that GeneChip® Hybridization Oven 645 is calibrated and set
to the correct temperature. Confirm that Stain 1 and Stain 2 are
placed in the correct order on the fluidics station. Ensure that Stain
Buffer 1 is stored in the dark when not in use. Use only those reagents
provided by Affymetrix.
One or more of the sub-grids in the
.dat file image were not gridded by
the software.
Follow the AGCC manual griding procedure to fix the grids and regenerate the CEL file.
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