CytoChip Oligo Reference Manual - Support

CytoChip Oligo Reference Manual - Support
CytoChip Oligo Microarray
Reference Guide
Revision History
Introduction
CytoChip Oligo Workflow
Sample Preparation
CytoChip Oligo Labeling
CytoChip Oligo Hybridization
CytoChip Oligo Washing
Scanning
Appendix
Technical Assistance
ILLUMINA PROPRIETARY
Part # 15056493 Rev. C
June 2015
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Revision History
Revision History
Part #
Revision
Date
15056493
C
June 2015
Updated hyperlinks and Technical Assistance page.
15056493
B
October 2014
Corrected wash buffer for wash 2 to Wash Buffer 2.
15056493
A
July 2014
CytoChip Oligo Microarray Reference Guide
Description of Change
Initial release
3
Introduction
CytoChip Oligo microarrays use array comparative genomic hybridization (array CGH)
approaches to investigate genomic copy number imbalance. Array CGH enables
independently labeled sample and control DNA to be competitively hybridized to
genomic probes, of known sequence, immobilized on a glass microarray. Software
analysis of the scanned microarray is used to estimate the amount of sample and control
DNA bound to each probe location. Analysis is also used to estimate the copy number of
the genomic sequence represented by that probe. This manual covers all stages of the
array CGH approach with CytoChip Oligo microarrays.
Experimental Design and Array Format
Single Hybridization – Multiple Samples per Slide
CytoChip Oligo format arrays use large numbers of features and software smoothing to
enable robust results to be reported. The results are based on a single hybridization of a
Cy3 labeled sample against a Cy5 labeled control.
The CytoChip Oligo arrays are multiformat so that a single slide supports multiple
samples. The CytoChip Oligo 2x105K array supports the analysis of 2 DNA samples on
each physical slide (8 DNA samples on 4 slides, 16 per pack of 8). CytoChip ISCA
4x44K, CytoChip ISCA 4x180K, CytoChip Oligo SNP 4x180K, and CytoChip Focus
4x180K support 4 DNA samples per slide (16 per pack of 4). CytoChip ISCA 8x60K and
CytoChip Focus 8x60K arrays support 8 DNA samples per slide (32 per pack of 4). Each
format provides a different resolution of coverage of the genome.
Array Design
The CytoChip Oligo 2x105K and CytoChip ISCA arrays have a constitutional diseasefocused design. The ICCG (International Collaboration for Clinical Genomics), committed
to establishing a uniform array format for use in clinical cytogenetics laboratories
worldwide, formulated the ISCA (International Standard Cytogenetic Array) design. For
more information on the ISCA design, refer to: Baldwin et al. (2008), Genetics in
Medicine, Jun;10(6):415-29 ihttp://www.ncbi.nlm.nih.gov/pubmed/18496225. The array
design includes attention to cytogenetically important regions such as telomeres,
centromeres, and pseudoautosomal regions. It also includes specific
microdeletion/microduplication syndromes and important Mendelian disease loci
associated with developmental delay and autism.
The CytoChip Focus array consists of a constitutional design for the investigation of
challenging samples, such as those with low amounts of poor quality DNA extracted
from CVS and amniocytes. This design is aimed to include genes associated with early
developmental disorders and specifically exclude late-onset diseases. The backbone is
replicated to provide more robust results while minimizing the reporting of Variants Of
Unknown Significance (VOUS). The CytoChip Focus 4x180K array can be used with only
200 ng of starting material,
Combined Array CGH and SNP Arrays
CytoChip Oligo SNP 4x180K array is a SNP array with a constitutional design. CytoChip
Oligo SNP consists of approximately 150,000 probes of the ISCA v2 4x180K targeted
design. In addition, 27,000 SNP-specific probes are present, distributed as evenly as
possible across the genome to provide approximately 10 Mb LOH resolution.
4
Part # 15056493 Rev. C
To improve the tracking of samples, especially on the more highly multiplexed formats,
Illumina provides CytoChip Spike-in Controls. Probe targets for spike-in controls have
been included on certain array designs and can be easily incorporated into the CytoChip
laboratory protocol.
Table 1 CytoChip Spike-in Controls available
Description
Quantity
Catalog No.
CytoChip Spike-in Controls
8 x 80 µl
PR-40-415301-00
CytoChip arrays are available in packs that provide all the reagents required to perform
an assay, including: CytoChip arrays, Fluorescent Labeling System [dUTP] (32 rxns) (PR30-413401-00), or SureLabel32SNP [dUTP] Fluorescent Labeling System (32 rxns) (PR-30413437-00), and COT Human DNA (PR-40-413503-00).
SureLabel32SNP [dUTP] Fluorescent Labeling System is provided specifically for use
with the CytoChip Oligo SNP array and includes: reagents for the digestion step,
characterized reference DNA (male and female) with a known SNP genotype, columns
for clean-up post labeling, and reagents for the fluorescent labeling procedure. The
genotype files required for SNP analysis with BlueFuse Multi are available for download
from www.cambridgebluegnome.com/account-login.
Table 2 CytoChip arrays available in packs
Description
Name
(AMADID/lot no.)
CytoChip Oligo 142 disease regions and a
2x105K Pack
30 Kb backbone (21856)
CytoChip ISCA 232 regions, 70 Kb
v1.0 4x44K Pack backbone (23097)
CytoChip ISCA 500 regions, 18 Kb
v1.0 4x180K
backbone (24585)
Pack
CytoChip ISCA 498 regions, 51 Kb
v2.0 8x60K Pack backbone (26370)
CytoChip ISCA 498 disease regions and a
v2.0 2x105K
30 Kb backbone (28740)
Pack
CytoChip ISCA 500 disease regions and a
v2.0 4x180K
21 Kb backbone (30078)
Pack
CytoChip ISCA 232 regions, 70 Kb
v2.0 4x44K Pack backbone (28739)
CytoChip Focus 162 severe early
8x60K Pack
disorders, 97 Kb
duplicated backbone
(40942)
CytoChip Focus 179 severe early
4x180K Pack
disorders, 44 Kb
triplicated backbone
(45830)
CytoChip Oligo 500 regions, 20 Kb
SNP 4x180K
backbone, 10 Mb LOH
Pack
resolution (33485)
CytoChip Oligo Microarray Reference Guide
No. of
samples
Spike-in
Compatible
16
No
PR-10-408001-PK
16
No
PR-10-408003-PK
16
No
PR-10-408006-PK
32
Yes
PR-10-408006-PK
16
Yes
PR-10-408011-PK
16
Yes
PR-10-408013-PK
16
Yes
PR-10-408010-PK
32
Yes
PR-10-408024-PK
16
Yes
PR-10-408026-PK
16
Yes
PR-11-448006-PK
Catalog No.
5
Introduction
CytoChip Spike-in Controls
User Supplied Reagents
Table 3 Reagents required, not provided
Reagent
Amicon Ultracel-30 membrane (columns included
in SNP Packs)
Commercial control DNA (100 ng/µl)
Aqueous glycogen solution
Oligo aCGH Wash Buffer 1 and 2
[Optional] CytoChip Spike-in Controls
[Optional] ClearPack Lite
6
Part Number
Millipore UFC503024 (24),
UFC503096 (96)
Promega G1521(female)/G1471
(male)
Sigma-Aldrich G1767
Illumina PR-70-413506-00
Illumina PR-40-415301-00
Illumina PR-70-431001-00
Part # 15056493 Rev. C
CytoChip Oligo protocols have been developed to fit into a standard laboratory workflow
as described in Figure 1. Protocols for CytoChip microarrays are performed within 2
days.
Figure 1 CytoChip Oligo Workflow Diagram
CytoChip Oligo Microarray Reference Guide
7
CytoChip Oligo Workflow
CytoChip Oligo Workflow
Sample Preparation
Ethanol precipitation is recommended to remove salts and other contaminants that
might lead to labeling problems. See DNA Quality on page 25 for further discussion on
when clean-up is required.
Materials
Table 4 Starting materials for sample preparation
Starting materials
Amount
DNA (unsheared, undigested, genomic DNA)
> 2 µg
Table 5 Materials required for sample preparation
Materials Required
Amount
Part Number
TE (10mM Tris, 1mM EDTA, pH7.0-8.0)
50 µl
Made up stock
Absolute ethanol
125 µl
70% ethanol
500 µl
Ultrapure water (Milli-Q, 18.2 MΩ.cm)
20 µl
Microcentrifuge tube (1.5 ml, flip cap)
Aqueous glycogen solution (1 µg/µl)
Sarstedt 72.690.001
2 µl
Prepared from Sigma
G1767
Clean Up DNA
NOTE
This step is optional.
1
Resuspend DNA in TE to a total volume of 50 µl.
2
Add 1/10th volume of 3M sodium acetate, vortex, then add 2.5 volume of absolute
ethanol. Invert twice to mix.
3
If starting with less than 2 µg of DNA, add 2 µl of aqueous glycogen solution
(1 µg/µl) as a coprecipitant.
4
Precipitate DNA for 2 hours at -25°C to -15°C.
NOTE
Alternatively, precipitation can be achieved in 30 minutes at -80°C.
8
5
Centrifuge at full speed (≥ 13,000 × g) for 15 minutes and decant the supernatant.
6
Add 500 µl of 70% ethanol and invert the tube three times to wash pellet.
7
Centrifuge at full speed (≥ 13,000 × g) for 5 minutes and decant the supernatant.
8
Pulse the tube in a centrifuge and remove the remaining ethanol with a P10 tip. Cap
tube.
Part # 15056493 Rev. C
Remove the cap and allow the pellet to air dry for 5 minutes at room temperature.
NOTE
Make sure that there is no residual ethanol visible around the pellet before
proceeding. Ethanol will inhibit subsequent labeling reactions.
10 Add 1x TE (pH 8.0) to give a final concentration of approximately 100 ng/µl.
11 Resuspend for 5 hours, or overnight, at room temperature.
12 Quantify DNA to confirm that OD readings are in line with recommendations (see
DNA Quality).
13 Proceed immediately to CytoChip Oligo Labeling or store DNA at 2°C to 8°C until
required.
CytoChip Oligo Microarray Reference Guide
9
Sample Preparation
9
CytoChip Oligo Labeling
Sample and reference DNAs are labeled with Cy3 and Cy5 fluorophores, respectively,
using random primers. Labeling mixes are combined, concentrated, and reconstituted to
the appropriate volume for hybridization.
To optimize dye incorporation using the fluorescent labeling system, include a restriction
digestion step before labeling. A restriction digestion step is highly recommended.
However, high-quality data can be achieved without digestion in certain circumstances.
By including a digestion step and running an agarose gel of the products, a good
impression of the DNA quality can be obtained that is independent of
spectrophotometric readings. If sample DNA is digested, it is also necessary to digest the
reference DNA to keep the DNA characteristics matched.
It is essential to perform restriction digestion of genomic DNA (sample and reference)
hybridized to the CytoChip Oligo SNP platform for the LOH/UPD calling to be effective.
Restriction digestion reagents are provided in the SureLabel32SNP [dUTP] Fluorescent
Labeling System.
CytoChip Oligo Spike-in Controls, if used, are incorporated into the CytoChip protocol at
the restriction digestion step or directly at the labeling step.
2x (CytoChip Oligo 2x105K) and 4x (CytoChip ISCA 4x44K and 4x180K, CytoChip Oligo
SNP 4x180K, and CytoChip Focus 4x180K) formats use the same quantities of starting
material and labeling reagents. The 8x (CytoChip ISCA 8x60K, CytoChip Focus 8x60K)
formats require half the quantity of labeled material; the 8x format is highlighted where
necessary.
Restriction Digestion of gDNA
Table 6 Starting materials for restriction digestion of gDNA
Starting Materials
Amount (2x and 4x
formats)
Amount CytoChip
Focus 4x180K
Amount (8x
formats)
Sample/Reference
DNA
1.0–1.5 µg in 20.2 µl
per hybridization
0.2–1.5 µg in 20.2 µl
per hybridization
0.4–0.5 µg in 10.1 µl
per hybridization
Sample DNA when
using CytoChip
Oligo Spike-ins
1.0–1.5 µg in 19.2 µl
per hybridization
0.2–1.5 µg in 19.2 µl
per hybridization
0.4–0.5 µg in 9.1 µl
per hybridization
Table 7 Reagents required for restriction digestion of gDNA
10
Reagents Required
Company and Part Number
PCR tube (0.2 ml, thin walled, flip cap) or 96well plate and adhesive seals
Thermo Scientific AB-0620 Thermo
Scientific AB-0600, AB-0558
Commercial control DNA (100 ng/µl)
Promega G1521(female)/G1471(male) or
SNP reference*
Alu I (10 U/µl)
Promega R6281 or provided in
SureLabel32SNP [dUTP]
Rsa I (10 U/µl)
Promega R6371 or provided in
SureLabel32SNP [dUTP]
Part # 15056493 Rev. C
Company and Part Number
10X buffer C
Supplied with Rsa I
OR 10X RE buffer
Supplied with SureLabel 32SNP [dUTP]
Acetylated BSA (10 µg/µl)
Supplied with Rsa I or with SureLabel
32SNP [dUTP]
Nuclease-free water
Supplied in labeling kit Illumina PR-30413401 or PR-30-413437-00
CytoChip Spike-in Controls (optional)**
Illumina PR-40-415301-00
*For SNP arrays, use a fully genotyped human reference DNA (provided with the SureLabel32SNP
[dUTP] Fluorescent Labeling System).
**CytoChip Oligo Spike-in Controls can be added at the digestion OR labeling step.
Restriction Digestion of gDNA Procedures
1
Thaw 10X buffer C or RE buffer and acetylated BSA. To collect contents, briefly
vortex and centrifuge. Retain reagents on ice while in use and return promptly to 25°C to -15°C.
2
For each reaction:
a Add the amount of genomic DNA to the appropriate nuclease-free tube or well in
the PCR plate.
b Add enough nuclease-free water to bring the final volume to 20.2 µl (2x and 4x
formats) or 10.1 µl (8x format).
c If using spike-ins controls, add enough nuclease-free water to bring the sample
volume to 19.2 µl (2x and 4x formats) or 9.1 µl (8x format).
3
If using spike-in controls:
a Remove a spike-in strip from the freezer and thaw on ice. Make sure that caps
are tightly closed.
b To collect contents, briefly vortex the strip and then centrifuge shortly. Orient the
strip with the cap hinge away from you.
c Accurately transfer 1 µl of Spike-in from each well to each tube or each well of
the 96-well plate containing the sample genomic DNA.
d Record the spike-in that has been added to each sample.
CAUTION
Add the sample tracking spike-ins to the sample DNA, do not add spike-ins to the
wells containing reference DNA.
CytoChip Oligo Microarray Reference Guide
11
CytoChip Oligo Labeling
Reagents Required
4
Prepare the Digestion Master Mix on ice in the order listed in Table 8. Briefly vortex
and centrifuge the Digestion Master Mix.
Table 8 Digestion Master Mix
Amount (2x
and 4x
formats)
Amount (2x
and 4x
formats)
1 rxn
16 rxns*
Nuclease-free
water
2.0 µl
10X buffer C or
RE buffer
Amount (8x
formats)
Amount (8x
formats)
1 rxn
16 rxns*
33.6 µl
1.0 µl
16.8 µl
2.6 µl
43.7 µl
1.3 µl
21.8 µl
acetylated BSA
0.2 µl
3.4 µl
0.1 µl
1.7 µl
Alu I (10U/µl)
0.5 µl
8.4 µl
0.25 µl
4.2 µl
Rsa I (10U/µl)
0.5 µl
8.4 µl
0.25 µl
4.2 µl
Final volume
5.8 μl
97.5 μl
2.9 μl
48.7 μl
Component
*master mix includes 5% excess
5
Add 5.8 µl (2x or 4x formats) or 2.9 µl (for 8x formats) of the Digestion Master Mix
to each reaction tube containing the genomic DNA. Make a total volume of 26 µl (2x
or 4x formats) or 13 µl (for 8x formats). Pipette to mix.
6
Transfer the samples to a thermal cycler and run the program in Table 9.
Table 9 Thermal cycler program for restriction digestion of gDNA
7
Step
Temperature
Time
1
37°C
2 hours
2
65°C
10 minutes
3
4°C
hold
Take 2 µl of the digested genomic DNA and run on a 0.8% agarose gel to assess the
completeness of the digestion. Make sure that most of the digested products are less
than 500 bp in length (see Agarose Gel Visualization of Digested gDNA on page 25).
a There will be 24 µl (2x or 4x formats) or 11 µl (8x formats) of digested genomic
DNA remaining.
b Add back the 2 µl as nuclease-free water to bring the volumes back to 26 µl and
13 µl in preparation for labeling.
NOTE
Complete digestion is essential for UPD/LOH calling to be effective with SNP
microarrays.
8
Proceed directly to Labeling (2x and 4x Formats) on page 13 or Labeling (8x formats) on
page 15. Digested gDNA can be stored for up to a month at -25°C to -15°C.
Labeling Materials
The materials listed are for 16 reactions, sufficient for 8 hybridizations.
12
Part # 15056493 Rev. C
Amount (2x and
4x formats)
Amount
CytoChip Focus
4x180K
Amount (8x
formats)
Sample/reference DNA
1.0–1.5 µg in 26
µl per
hybridization
0.2–1.5 µg in 26
µl per
hybridization
0.4–0.5 µg in 13
µl per
hybridization
Sample DNA when using
CytoChip Oligo Spike-ins
1.0–1.5 µg in 25
µl per
hybridization
0.2–1.5 µg in 25
µl per
hybridization
0.4–0.5 µg in 12
µl per
hybridization
Starting Materials
Table 11 Materials required for labeling
Materials Required
Amount (2x and
4x formats)
Amount (8x
formats)
Commercial control
DNA* (100 ng/µl)
0.2–1.5 µg per
hybridization
0.4–0.5 µg per
hybridization
Promega G1471/G1521
PCR tube (0.2 ml, thin
walled, flip cap) or 96well plate and
adhesive seals
16 PCR tubes or
1 96-well plate
16 PCR tubes
or
1 96-well plate
Thermo Scientific AB0620
Thermo Scientific AB0600, AB-0558
Fluorescent Labeling
System [dUTP]
Half a kit
Quarter of a
kit
Illumina PR-30-413401/
PR-30-413437-00 or in
pack
Amicon Ultracel-30
membrane (columns
included in SNP Packs)
16
16
Millipore UFC503024
(24), UFC503096 (96)
TE (pH 8.0)
16 ml
16 ml
Microcentrifuge tube
(1.5 ml, flip cap)
50
50
Sarstedt 72.690.001
CytoChip Spike-in
Controls (optional)**
1 µl per sample
1 µl per
sample
Illumina PR-40-4153000
Part Number
*For the control, match the DNA quantity used in the sample - CytoChip Focus 4x180K can be as low
as 200 ng.
**CytoChip Oligo Spike-in Controls can be added at the digestion OR labeling step.
For more information on required materials, see Labeling Notes on page 25.
Labeling (2x and 4x Formats)
NOTE
Perform all steps on ice unless otherwise indicated.
1
Thaw components from Fluorescent Labeling System, and then vortex briefly. To
collect contents, centrifuge the components. Retain on ice.
CytoChip Oligo Microarray Reference Guide
13
CytoChip Oligo Labeling
Table 10 Starting materials for labeling
2
If using spike-in controls, remove a spike-in strip from the freezer and thaw on ice.
Make sure that caps are tightly closed.
a Briefly vortex the strip followed by a short centrifugation. Orient the strip with
the cap hinge away from you.
b Accurately transfer 1 µl of spike-in from each well to each tube or each well of
the 96-well plate containing the sample genomic DNA.
c Record the spike-in that was added to each sample.
CAUTION
Add the sample tracking spike-ins to the sample DNA, do not add spike-ins to the
wells containing reference DNA.
3
Add 5 µl of random primers to each reaction tube containing 26 µl of gDNA to make
a total volume of 31 µl. Pipette up and down gently to mix. Centrifuge to collect the
contents.
4
Transfer samples to a prewarmed lidded thermal cycler at 95°C for 5 minutes (if
restriction digestion used) or for 10 minutes (if no restriction digestion is used). Then
transfer immediately to ice or to a precooled thermal cycler for 5 minutes at 4°C.
NOTE
Rapid cooling of the denatured labeling mixes is critical for high efficiency labeling.
Poor cooling can be associated with increased dye bias in GC-rich regions, for
example 1 p and chromosomes 19 and 22.
5
Centrifuge the samples for 1 minute at 6,000 × g to collect the contents at the bottom
of each tube. Centrifuge PCR plates at 170 × g.
6
Prepare the labeling master mixes by adding the components in the quantities and
order listed in Table 12.
Table 12 Labeling master mix components and quantities (2x and 4x formats)
Cy3
labeling
mix
Cy3
labeling
master mix
Cy5
labeling
mix
Cy5
labeling
master mix
1 rxn
8 rxns*
1 rxn
8 rxns*
5x Reaction buffer
10 µl
84 µl
10 µl
84 µl
10x dNTP
5 µl
42 µl
5 µl
42 µl
Cy3 dUTP
3 µl
25.2 µl
3 µl
25.2 µl
Component
Cap
color
Cy5 dUTP
Exo-Klenow fragment
1 µl
8.4 µl
1 µl
8.4 µl
Total
19 μl
159.6 μl
19 μl
159.6 μl
*Labeling master mix includes 5% excess.
7
14
Add 19 µl of the labeling master mix to each reaction tube to make a total volume of
50 µl. Cy3 mix is added to the sample gDNA and Cy5 is added to the reference.
Gently pipette up and down to mix. Centrifuge to collect the contents.
Part # 15056493 Rev. C
Transfer the samples to a thermal cycler and run the program in Table 13.
Table 13 Thermal cycler program for labeling (2x and 4x formats)
9
Step
Temperature
Time
1
37°C
2 hours
2
65°C
10 minutes
3
4°C
hold
Proceed directly to Combination on page 16. Alternatively, reactions can be stored up
to a month at -25°C to -15°C in the dark.
Labeling (8x formats)
NOTE
Perform all steps on ice unless otherwise indicated.
1
Thaw components from Fluorescent Labeling System, vortex briefly. Centrifuge to
collect the contents. Retain on ice.
2
If using spike-in controls, remove a spike-in strip from the freezer and thaw on ice.
Make sure that caps are tightly closed.
a Briefly vortex the strip, then briefly centrifuge. Orient the strip with the cap hinge
away from you.
b Accurately transfer 1 µl of spike-in from each well to each tube or each well of
the 96-well plate containing the sample genomic DNA.
c Record the spike-in that was added to each sample.
CAUTION
Add the sample tracking spike-ins to the sample DNA, do not add spike-ins to the
wells containing reference DNA.
3
Add 2.5 µl of random primers to each reaction tube containing 13 µl of gDNA to
make a total volume of 15.5 µl. Pipette up and down gently to mix. Centrifuge to
collect the contents.
4
Transfer samples to a prewarmed lidded thermal cycler at 95°C for 5 minutes (if
restriction digestion used) or for 10 minutes (if no restriction digestion is used). Then
transfer immediately to ice or to a precooled thermal cycler for 5 minutes at 4°C.
NOTE
Rapid cooling of the denatured labeling mixes is critical for high efficiency labeling.
Poor cooling can be associated with increased dye bias in GC-rich regions, for
example 1 p and chromosomes 19 and 22.
5
Centrifuge the samples for 1 minute at 6,000 × g to collect the contents at the bottom
of each tube. Centrifuge PCR plates at 170 × g.
6
Prepare the labeling master mixes by adding the components in the quantities and
order listed in Table 14.
CytoChip Oligo Microarray Reference Guide
15
CytoChip Oligo Labeling
8
Table 14 Labeling master mix components and quantities (8x formats)
Cy3 labeling
mix
Cy3 labeling
master mix
Cy5 labeling
mix
Cy5 labeling
master mix
1 rxn
8 rxns*
1 rxn
8 rxns*
5x Reaction
buffer
5 µl
42 µl
5 µl
42 µl
10x dNTP
2.5 µl
21 µl
2.5 µl
21 µl
Cy3 dUTP
1.5 µl
12.6 µl
1.5 µl
12.6 µl
Component
Cap
color
Cy5 dUTP
Exo-Klenow
fragment
0.5 µl
4.2 µl
0.5 µl
4.2 µl
Total
9.5 μl
79.8 μl
9.5 μl
79.8 μl
*Labeling master mix includes 5% excess.
7
Add 9.5 µl of the labeling master mix to each reaction tube to make a total volume of
25 µl. Cy3 mix is added to the sample gDNA and Cy5 mix is added to the reference.
Gently pipette up and down to mix. Centrifuge to collect the contents.
8
Transfer the samples to a thermal cycler and run the program in Table 15.
Table 15 Thermal cycler program for labeling (8x formats)
9
Step
Temperature
Time
1
37°C
2 hours
2
65°C
10 minutes
3
4°C
hold
Proceed directly to Combination. Alternatively, reactions can be stored up to a month
at -25°C to -15°C in the dark.
Combination
The labeled genomic DNA is cleaned up using Amicon Ultracel-30 membrane filters
(AU-30), purchased separately or provided with SureLabel32SNP [dUTP], followed by
vacuum concentration if necessary. Labeled sample and reference genomic DNA is
combined for each hybridization area. If many samples are being processed
simultaneously, use a 96-well plate format for more convenient processing. See
www.chem.agilent.com (part G4410-90010) for the recommended use of AutoScreen-96A
Well plates (GE Healthcare p/n 25- 9005- 98).
16
1
Centrifuge the labeled genomic DNA samples for 1 minute at 6,000 × g to collect the
contents at the bottom of each tube, or at 170 × g for PCR plates. Transfer each
sample or reference to a separate 1.5 ml microcentrifuge tube.
2
Add 430 µl of 1x TE (pH 8.0) to each reaction tube.
3
For each sample or reference, place an AU-30 filter into a collection tube (supplied
with Amicon filters) and load each labeled gDNA into the filter. Centrifuge at 14,000
× g at room temperature for 10 minutes, discard flow through.
Part # 15056493 Rev. C
Return the filter to the collection tube, add 480 µl of 1x TE (pH 8.0) to each filter,
centrifuge at 14,000 × g at room temperature for 10 minutes, discard flow through.
5
Invert the filter into a fresh collection tube (supplied), centrifuge at 1,000 × g at room
temperature for 1 minute to collect the purified sample.
6
Measure the volume collected with a pipette. There will be approximately 21 µl, add
1x TE, or use a vacuum concentrator to bring the sample to the volume required:
Table 16 Sample volume required for combination
2x105K
Collected Volume
21 µl
1x TE
20 µl
41 μ
Volume required
7
4x Format
8x60K
21 µl
21 μl
21 µl
9.5 μl
For the 8x60K formats, concentration of the sample is required. Place sample tubes in
a vacuum concentrator (prewarmed to 75°C or higher) with tops open and evaporate
to dryness (~20–40 minutes). Then reconstitute with 9.5 µl of 1x TE. If no vacuum
concentrator is available, ethanol precipitation can be used (see Ethanol Precipitation
of Labeling on page 26).
NOTE
With a miVAc Duo (Genevac) fitted with a swing rotor for microplates (Genevac
#DRS-00000-200), evaporation is completed in 40–45 minutes at 75°C.
8
Take 1.5 µl of each sample and use a NanoDrop Spectrophotometer to determine
DNA concentration, dye incorporation, and specific activity for each sample (see
Recording DNA Yield and Dye Incorporation on page 27).
9
Combine the Cy3 labeled sample and the Cy5 labeled reference DNA for each
hybridization area.
10 Proceed to CytoChip Oligo Hybridization. Alternatively, reactions can be stored up to a
month at -25°C to -15°C in the dark.
CytoChip Oligo Microarray Reference Guide
17
CytoChip Oligo Labeling
4
CytoChip Oligo Hybridization
This section describes combination of the labeled DNA with blocking agents and
Hybridization Buffer, followed by hybridization using Agilent hybridization chambers.
Before proceeding with Hybridization, prepare the 10X blocking agent. Add 900 µl of
nuclease-free water to the vial containing the lyophilized 10X blocking agent. Leave the
vial at room temperature for 1 hour. Vortex before use or store the vial at -25°C to -15°C.
For more comprehensive guidance on loading samples and assembly/disassembly of
Agilent Microarray Hybridization Chambers, refer to the Agilent Microarray
Hybridization Chamber User Guide (G2534-90001).
NOTE
The “Agilent”-labeled barcode is on the active side of the slide.
Materials
Table 17 Starting materials for hybridization
Starting Material
8 hybs on 4
2x format
slides
8 hybs on 2
4x format
slides
8 hybs on 1
8x format
slide
Part number, or
included in
Packs
COT Human DNA
200 µl
40 µl
16 µl
PR-40-413503-00
2X Hi-RPM buffer
1040 µl
440 µl
180 µl
Supplied with
Labeling Kit
10X blocking agent
(reconstituted)
208 µl
88 µl
36 µl
Supplied with
Labeling Kit
CytoChip Oligo 2x105K
slides and gasket slides
4
CytoChip ISCA 4x44K
slides and gasket slides
2
PR-21-40800300/ PR-21408010-00
CytoChip ISCA 4x180K
slides and gasket slides
2
PR-21-40800600/ PR-21408013-00
CytoChip ISCA 8x60K
slides and gasket slides
1
PR-21-408005-00
CytoChip Focus 8x60K
slides and gasket slides
1
PR-21-408024-00
CytoChip Focus 4x180K
slides and gasket slides
2
PR-21-408026-00
CytoChip Oligo SNP
4x180K slides and gasket
slides
2
PR-23-438006-00
Agilent Microarray
Hybridization Chambers
18
PR-21-408001-00
4
2
1
Agilent G2534A
Part # 15056493 Rev. C
Preparation
1
Prewarm a hotblock to 95°C.
2
Prewarm the Agilent Hybridization Oven to 65°C.
3
Prewarm a heat block or waterbath to 37°C.
Hybridization
1
To each tube containing labeled DNA, add COT, blocking agent, and Hybridization
Buffer according to Table 18. To collect the contents, mix and pulse centrifuge each
tube.
Table 18 CytoChip hybridization reagents
Volume per
2x105K
hybridization
Volume per 4x
format
hybridization
Volume per
8x60K
hybridization
Labeled DNA solution (test and
reference combined)
79 µl
39 µl
16 µl
COT Human DNA
25 µl
5 µl
2 µl
10X blocking agent
26 µl
11 µl
4.5 µl
2X Hybridization Buffer (Hi-RPM)
130 µl
55 µl
22.5 µl
Total
260 μl
110 μl
45 μl
Reagent
2
Incubate for 3 minutes at 95°C in a prewarmed hotblock. Transfer immediately to a
prewarmed hotblock or waterbath at 37°C, and incubate for a further 30 minutes.
Cool to room temperature and pulse centrifuge.
3
Load a clean gasket slide of the correct format (2x, 4x, or 8x) into an Agilent
Microarray Hybridization Chamber base with the gasket label facing upwards over
the rectangular alignment area.
4
Using a “drag and dispense” method, slowly dispense 245 µl (2x105K), 100 µl (4x
formats), or 40 µl (8x60K) of labeled hybridization mixture onto a gasket well. For
more information, see Loading Hybridization Solution on page 28.
a To prevent leakage, avoid contacting the o-ring of the gasket slide with the
hybridization mixture.
b Load hybridization mixture into all gasket wells before applying the CytoChip
Oligo slide. See Multiformat Subarray Layout on page 29 for the naming of
subarrays on the different CytoChip Oligo formats.
5
Place a CytoChip Oligo slide array-side down onto the gasket slide, with the numeric
barcode facing upwards and the Agilent-labeled barcode downwards. The Agilentlabeled side is the active side of the array. Make sure that the slide and gasket are
aligned (see Assembly of Agilent Hybridization Chamber on page 29).
CytoChip Oligo Microarray Reference Guide
19
CytoChip Oligo Hybridization
CAUTION
Microarray slides and gasket slides are made of glass. Handle with care. Inspect the
slides for any damage or imperfections before removing from the packaging to make
sure that no damage has occurred in transit.
6
Place the hybridization chamber cover onto the chamber base. Slide the clamp
assembly onto the chamber and hand-tighten.
7
Holding the assembled chamber vertically (see Figure 2), rotate clockwise 3 times to
wet the slides and assess the mobility of the bubbles (see Quality of Bubbles). If
necessary, tap the chamber on a hard surface to move stationary bubbles.
Figure 2 Assembled hybridization chamber
20
8
Place the assembled slide chamber in the rotator rack in a prewarmed Hybridization
Oven at 65°C. Rotate at 20 rpm and hybridize at 65°C for 24 hours (4x formats or
8x60K), or 40 hours (2x105K).
9
Proceed with CytoChip Oligo Washing.
Part # 15056493 Rev. C
Hybridized CytoChip Oligo slides are washed to remove unhybridized labeled DNA.
This protocol uses a ClearHyb Wash System. If a ClearHyb Wash System is not
available, see CytoChip Oligo Oven-Based Wash on page 31. For general guidance on wash
conditions, see Additional Wash Notes on page 32. Cover the room temperature wash in
foil to keep dark.
Materials and Equipment
Prepare the following buffers:
Table 19 CytoChip Oligo wash buffers
Wash
Volume
Temp
Disassembly
400 ml
RT
1
400 ml
RT
2
500 ml
37 °C
Times
Agitation
Buffer
None
CytoChip Oligo aCGH Wash Buffer
1, Illumina PR-70-413506-00
5 min
2.5 cm
stir bar
CytoChip Oligo aCGH Wash Buffer
1, Illumina PR-70-413506-00
1 min
None
CytoChip Oligo aCGH Wash Buffer
1, Illumina PR-70-413506-00
Prepare the following equipment:
Table 20 CytoChip Oligo wash equipment
Item
Number
Comments
400 ml lidded square glass staining
dishes
2
Use for disassembly and wash 1.
ClearHyb Wash System
1
Pre-fill the waterbath with CytoChip Oligo
aCGH Wash Buffer 1 preheated to 37 °C for
30 minutes in preparation for wash 2.
25 position stainless steel staining
rack
1
Place the rack in wash 1
Magnetic stirrer
1
2.5 cm stir bar
1
NOTE
Glass containers are recommended for washing. For room temperature washes, use
foil covers over glass jars to protect slides from light.
NOTE
Calibrate the ClearHyb unit following the manufacturer instructions. When the
ClearHyb is calibrated, recheck the temperature using a calibrated thermometer
before each wash.
Wash Procedures
1
Disassemble hybridization chambers on the bench (see Disassembly of Agilent
Hybridization Chambers on page 32).
CytoChip Oligo Microarray Reference Guide
21
CytoChip Oligo Washing
CytoChip Oligo Washing
a
b
c
22
Submerge the gasket and microarray slide in the CytoChip Oligo aCGH Wash
Buffer 1 in a square glass staining dish.
Separate the gasket slide from the microarray.
Transfer the CytoChip Oligo microarrays to the slide rack in wash 1. Only touch
the edge or barcode of the slide.
2
When the rack is fully loaded (wash no more than 12 slides at a time), replace glass
lid, switch on stirrer and adjust for good, but not vigorous, stirring. Cover with foil
and stir for 5 minutes at room temperature.
3
Transfer the slide rack to the ClearHyb containing prewarmed CytoChip Oligo aCGH
Wash Buffer 1 at 37°C, and incubate for 1 minute.
4
Optional: In a high-ozone environment, use the Ozone-Barrier slide cover kit supplied
by Agilent (G2505-60550). The Ozone-Barrier slide cover protects Cy dyes from ozone
degradation.
5
Slowly lift the slide rack out of the ClearHyb, allowing the liquid to draw droplets off
the microarray surface. It takes 10–12 seconds to remove the slide rack. Store the
slides in the original packaging.
6
Proceed with Scanning.
Part # 15056493 Rev. C
A laser scanner is used to excite the hybridized fluorophores and record the resulting
images of the hybridization. A two channel scanner is required to image the Cy3 and
Cy5 signals produced by the independently labeled sample and control DNA. The
resulting images are stored in TIFF format file, which the BlueFuse Multi analysis
software reads.
Any 2 channel laser scanner equipped with the following lasers is able to scan a
CytoChip Oligo slide.
} Green laser, 532 nm wavelength – used to excite and read the Cy3 signal.
} Red laser, 635 nm wavelength – used to excite and read the Cy5 signal.
White light, CCD based, scanners can also be used. However, such devices are likely to
produce poorer results, which are more difficult to interpret and report.
Although there some laser scanners available from commercial suppliers, they all share
a basic workflow.
NOTE
2x105K and 4x44K formats require 5 µm scanning resolution.
4x180K and 8x60K formats require 3 µm scanning resolution.
Illumina does not support 2 µm scanning.
Prescan
CytoChip oligos have 2 (2x105K), 4 (4x44K, 4x180K) or 8 (8x60K) hybridization areas.
The prescan provides a low-resolution image of the entire slide, which is used to identify
the hybridization areas. Most scanners also read and interpret the barcode of the slide
during the prescan, which can be used when saving the file.
For some scanners, such as those supplied by Agilent, it can be more convenient to scan
the whole slide at higher resolution. The image can then be split using Image Viewer
software available for download at www.cambridgebluegnome.com/account-login.
Alternatively, images can be split during batch import into BlueFuse Multi.
Scan Resolution
Typically scans are produced from 2 to 10 µm resolution. High-resolution scans of 5 µm
(2x105K, 4x44K) and 3 µm (4x180K, 8x60K) are recommended for CytoChip Oligo
arrays.
Saving of TIFF images
To simplify the workflow, it is recommended that the following settings are used when
saving TIFF images (see Figure 3).
} Place the barcode within the first or second string of the file name. For example,
158028A_S01_TOP.tif or US85103613_158028A_S01_TOP.tif, and NOT rimilab_201007-16_12h38m_158028A_CY3-34627_CY5-34759_TOP.tif.
} Store all the scans, potentially 2 prescans (Cy3 and Cy5) and 2 high-resolution scans,
in a single, multiframe TIFF file.
} Use a consistent suffix, such as “top” and “bottom”, to identify the hybridization
area that is included in the file.
CytoChip Oligo Microarray Reference Guide
23
Scanning
Scanning
Figure 3 Saving TIFF images
If these recommendations are followed, the BlueFuse Multi Software automatically loads
the resulting images, saving time and minimizing a potential source of error.
Proceed with Data Analysis. Refer to the BlueFuse Multi Walkthrough guide.
24
Part # 15056493 Rev. C
Appendix
Appendix
DNA Quality
Confirm that the unsheared, undigested, genomic DNA is:
High molecular weight
Protein/RNA free
Solvent free
> 10–20 kbp
OD 260/280 > 1.8
OD 260/230 > 2.0
As an additional quality check, run the DNA on a 0.8–1% agarose gel (seeFigure 4). DNA
of high molecular weight appears as a series of tight bands towards the middle of gel
(marked in green). Fragmented DNA, with a mixture of molecular weights appear as a
vertical smear (marked in red on left). Impurities appear as small bands at the top
(marked in red at top). Overloading of the gel can result in overly bright bands, which
make it difficult to interpret the gel correctly (marked in yellow).
Figure 4 DNA that is run on 1% agarose gel
DNA that fails to meet these quality criteria can be prepared using this protocol, which
describes ethanol precipitation, to reduce salt contamination.
Store NanoDrop readings, gel images, and extraction protocols in the sample record of
the BlueFuse Multi analysis software.
Labeling Notes
Agarose Gel Visualization of Digested gDNA
Figure 5 Examples of good gDNA digestion and poor gDNA digestion
Fully digested gDNA is visible on an agarose gel with most fragments sized less than
500 bp, as shown on the left gel in Figure 5. Poorly digested samples, shown on the right
gel in Figure 5, have a continuous smear of DNA, with no significant size concentration
of fragments. Full digestion is imperative for effective LOH calling when using SNP
arrays. The digested samples from the gel on the right did not perform well on CytoChip
Oligo SNP arrays.
CytoChip Oligo Microarray Reference Guide
25
gDNA Input Amount and Volume per Microarray
At least 1.0 µg of genomic DNA is required in labeling reactions for 2x105K or 4x
formats (0.4 µg minimum is required for 8x60K format). Equal amounts of genomic
DNA are required for both the experimental and reference channels.
Table 21 gDNA input amount and volume per microarray
Volume of gDNA
gDNA input
with restriction
Microarray format
requirement (μg)
digestion (μl)
2x105K or 4x formats
1.0 to 1.5
20.2
8x60K
0.4 to 0.5
10.1
Volume of gDNA
without restriction
digestion (μl)
26
13
Choice of Reference DNA
If the sex of the sample is known, selection of reference DNA is based on the sex of the
sample. In general, sex-mismatched designs are used when array CGH approaches are
first implemented because the offset of the X chromosome provides useful information
on assay performance. Sex-matched designs are more commonly used when consistent
results are obtained. If the sex of the sample is unknown, use a dyeswap design with one
hybridization undertaken with male control DNA and the other undertaken with female
control DNA.
For standard CytoChip Oligo arrays, commercial pooled DNA is recommended.
However, there is evidence that results are improved when sample and control DNA are
extracted using the same technique.
SNP platforms require a single well-characterized reference with a known SNP genotype.
Do not pool reference DNA. Illumina supplies SureLabel32SNP [dUTP] with SNP arrays
that includes SNP reference DNA.
Component Quantities for Single Reactions
The Fluorescent Labeling System is supplied in a 32 reaction format optimized to
produce master mixes for 16 reactions for each Cy dye. Take care when making master
mixes for smaller numbers of reactions, particularly with Cy dyes, so that there are
sufficient reagents to complete the total of 16 reactions. For example, a volume of 0.8
µl/hour is lost to evaporation from an open tube.
Ethanol Precipitation of Labeling
For the 8x60K array format, it is necessary to reduce the volume of the labeled samples
after cleaning up with the AU-30 filters. A vacuum concentrator is recommended
However, there is an alternative method using ethanol precipitation. First check the DNA
yield and specific activity using a NanoDrop spectrophotometer (and record in BlueFuse
Multi if necessary). Then combine the labeled sample and reference together with the
COT human DNA before performing the ethanol precipitation. This step improves the
DNA recovery.
NOTE
If the volumes recovered from the Amicon columns are greater than 22 µl, it is also
necessary to concentrate the samples for 4x format. Therefore volumes are also
included here for 4x formats in bold.
26
Part # 15056493 Rev. C
NOTE
Without restriction digestion, the specific activity values will be about 2 pmol/µg
lower than values indicated in this table.
1
Combine ~21 µl of labeled test, ~21 µl of labeled reference with 2 µl (5 μl) of COT
human DNA.
2
Add 5.5 µl of sodium acetate, mix thoroughly and add 150 µl of 100% ethanol.
Invert to mix.
3
Precipitate for 2 hours at -25°C to -15°C (recommended). Alternatively, precipitation
can be achieved in 30 minutes at -80°C.
4
Centrifuge at full speed (≥ 13,000 × g) for 15 minutes and decant supernatant
5
Add 300 µl of 70% ethanol and invert the tube three times to wash the pellet.
6
Centrifuge at full speed (≥ 13,000 × g) for 5 minutes and decant supernatant.
7
Pulse tube in centrifuge and remove remaining ethanol with a P10 tip. Cap tube.
8
Remove cap and allow the pellet to air dry for 5 minutes at room temperature. Make
sure that there is no residual ethanol visible around the pellet before proceeding as
ethanol will inhibit subsequent labeling reactions.
9
Resuspend in 18 µl (44 μl) of 1x TE (pH 8.0).
10 Return to Hybridization on page 19. At step 1, there is no need to add COT.
Recording DNA Yield and Dye Incorporation
Use the NanoDrop 2000 UV-VIS Spectrophotometer or equivalent to measure the yield,
degree of dye incorporation of labeling reactions.
1
From the main menu, select MicroArray Measurement, then from the Sample Type
menu, select DNA-50.
2
Use 1.5 µl of TE to blank the instrument.
3
Use 1.5 µl of purified labeled genomic DNA for quantification. Measure the
absorbance at A260nm (DNA), A550nm (cyanine 3), and A650nm (cyanine 5).
4
Record the dye incorporation (pmol dye/µl) for Cy3 or Cy5 for each sample or
reference.
5
Record the gDNA concentration (ng/µl) for each sample.
6
Calculate the specific activity of your sample:
Specific activity (pmol/µg) =
7
dye incorporation pmol per µl dye
DNA concentration µg per µl genomic DNA
Acceptable values, after labeling and clean-up:
CytoChip Oligo Microarray Reference Guide
27
Appendix
Table 22 Acceptable values for DNA yield and dye incorporation after labeling and clean-up.
Values are based on the volumes recovered from the clean-up step from 500 ng of starting
material.
Cy3
Specific
Cy5
Specific
Concentration
Volume
incorporation
activity Cy3 incorporation
activity Cy5
(ng/μl)
(pmol/μl)
(pmol/μg)
(pmol/μl)
(pmol/μg)
21 µl
117 to 167
3–6.8
>11.3
2.2–5.9
>9
25 µl
98 to 140
2.6–5.7
>9.5
1.9–5.0
>7.6
Table 23 Acceptable values if vacuum concentrator is used
Specific
Cy3
CytoChip
Concentration
activity
incorporation
(ng/μl)
Cy3
Oligo array
(pmol/μl)
(pmol/μg)
8x60K
260 to 370
7–15
>25
4x44K,
240 to 330
6–13
>25
4x180K
2x105K
120 to 170
3–7
>25
5–13
5–12
Specific
activity
Cy5
(pmol/μg)
>20
>20
2–6
>15
Cy5
incorporation
(pmol/μl)
NOTE
Without restriction digestion, the specific activity values are about 5 pmol/µg lower
than values indicated in Table 23.
8
Record DNA yield and dye incorporation for each reaction or save NanoDrop file for
later import into the BlueFuse Multi analysis software.
NOTE
If a NanoDrop instrument is not available, use other spectrophotometers (eg, Epoch
BioTek Gen 5). Measure the Cy3 incorporation by taking the absorbance at 553 nm
and, after removing the value of the blank, divide by 0.015 to give pmol/µl. Measure
the Cy5 incorporation by taking the absorbance at 649 nm and, after removing the
value of the blank, divide by 0.025 to give pmol/µl.
Hybridization Notes
Loading Hybridization Solution
Load the hybridization solution onto the appropriate gasket slide for the array format.
With the gasket slide already in the hybridization chamber, slowly dispense your sample
while moving your pipette to the opposite end of the well without touching the slide. Use
a “drag and dispense” pipetting method (see Figure 6).
Figure 6
Drag and dispense method
To avoid leakage of the hybridization solution, avoid touching the gasket with the
pipette. Dispense the solution in the center of the gasket (see Figure 7).
Figure 7 Correct placement of hybridization solution in gasket slides
NOTE
28
Part # 15056493 Rev. C
Multiformat Subarray Layout
The 2 subarrays of the CytoChip Oligo 2x105K microarray are named top and bottom by
the image viewer software/BlueFuse Multi batch import process.
Similarly, the 4 subarrays of the 4x array formats are labeled A-D, and the 8x60K format
subarrays are labeled A-H. Gasket view is how the gasket slide looks as you apply the
samples and slide view is how the slide looks on screen after scanning (see Figure 8).
Figure 8 Gasket View and Slide View
Assembly of Agilent Hybridization Chamber
1
When all gaskets are loaded with the correct hybridization solution, retrieve the
CytoChip Oligo slide. With the numeric barcode facing up, carefully lower on top of
the gasket slide.
Figure 9 CytoChip Oligo placed in hybridization chamber gasket slide
2
Place the chamber cover, correct side facing up, onto the chamber base containing
both slides.
3
Slip the clamp assembly onto the chamber base and cover until it stops firmly in
place. Tighten the thumbscrew on the completed chamber assembly.
CytoChip Oligo Microarray Reference Guide
29
Appendix
These images are for demonstration purposes only. Load gasket slides in the
hybridization chamber before loading the hybridization solution.
Figure 10 Hybridization chamber and clamp assembly
4
Rotate the final assembled chamber 2 to 3 times to wet the gaskets and to make sure
that the hybridization solution will coat the entire surface of the microarray during
the incubation process. Assess the mobility of the bubbles.
Figure 11 Assembled hybridization chamber
Quality of Bubbles
A single, large mixing bubble that freely moves around the chamber when rotated is best.
Multiple bubbles are acceptable as long as they move when you rotate the chamber. If
necessary, remove stationary bubbles by gently tapping each corner of the chamber
assembly on a firm surface.
Figure 12 Examples of bubbles in hybridization chamber
30
Part # 15056493 Rev. C
Appendix
Washing Notes
CytoChip Oligo Oven-Based Wash
Prepare the following buffers
Table 24 CytoChip Oligo oven-based wash buffers
Wash
Volume
Temp
Disassembly
400 ml
RT
1
400 ml
RT
2
500 ml
37 °C
Times
Agitation
Buffer
None
CytoChip Oligo aCGH Wash Buffer
1, Illumina PR-70-413506-00
5 min
2.5 cm
stir bar
CytoChip Oligo aCGH Wash Buffer
1, Illumina PR-70-413506-00
1 min
None
CytoChip Oligo aCGH Wash Buffer
2, Illumina PR-70-413506-00
Prepare the following equipment
Table 25 CytoChip Oligo oven-based wash equipment
Item
Number
Comments
400 ml lidded square glass staining dish
1
Prewarmed to 37 °C in an oven (2
hrs)*
400 ml lidded square glass staining dish
1
For disassembly
25 position stainless steel staining rack
1
Magnetic stirrer
2
2.5 cm stir bar
1
One prewarmed to 37 °C in an
oven (2 hrs)*
*For optimal stringency it is important that CytoChip Oligo microarrays are washed in oligo aCGH
Wash Buffer 2 at 37°C. Prewarm the buffer, staining dish, and magnetic stirrer in a calibrated oven
for 2 hours.
1
Disassemble the hybridization chambers on the bench. Separate the gasket slide from
the microarray in 400 ml square glass staining dish. Transfer the CytoChip Oligo
microarrays to the slide rack in wash 1 touching only the edge or barcode of the slide
(see Disassembly of Agilent Hybridization Chambers on page 32).
2
When rack is fully loaded, replace lid and protect from light with a foil cover. Switch
on stirrer and adjust for good, but not vigorous, stirring. Stir for a further 5 minutes
at room temperature. Meanwhile, prepare wash 2.
3
Prepare wash 2.
a Working quickly to keep materials warm, empty the water from the prewarmed
square glass dish.
b Add a 2.5 cm stir bar and place on the magnetic stirrer in the oven at 37°C.
c Pour 400 ml of prewarmed CytoChip Oligo aCGH Wash Buffer 2 into the dish
and replace the lid.
d Close the oven door to keep the materials at 37°C.
CytoChip Oligo Microarray Reference Guide
31
4
Transfer slide rack to wash 2 in oven for 1 minute.
5
Optional: In a high-ozone environment, use the Ozone-Barrier slide cover kit supplied
by Agilent (G2505-60550). The Ozone-Barrier slide cover is a highly effective way of
protecting Cy dyes from ozone degradation.
6
Slowly lift the slide rack out wash 2, allowing the liquid to draw droplets off the
microarray surface. It takes 5–10 seconds to remove the slide rack. Store slides in
original packaging.
Additional Wash Notes
} Complete all room temperature washes in dishes covered with silver foil.
} Always keep the rack of CytoChips in buffer. Transfer the rack of CytoChips
immediately after the completion of each wash.
} To reduce risk of contact between the stir bar and the array surface, place the
CytoChips in the rack facing away from the stir bar.
} When using a stirred wash, the rack holds the CytoChips above the stir bar. A vortex
is visible, but the CytoChips are always covered by liquid.
Disassembly of Agilent Hybridization Chambers
1
Remove 1 hybridization chamber from the incubator and place on a flat surface.
a Loosen the thumbscrew and slide the clamp off the assembly.
b Remove the chamber cover.
c With gloved fingers, hold the slide/gasket pair by the short ends and remove
from the chamber base.
d Submerge and hold in the disassembly bath with the numeric barcode of the
CytoChip Oligo facing upwards.
Figure 13 Slide and gasket submerged in the disassembly bath
2
32
Keeping the slide/gasket pair submerged, pry the sandwich open at the barcode end
using plastic forceps.
a Allow the gasket slide to drop to the bottom of the dish.
b Minimizing exposure to air, quickly transfer the CytoChip Oligo microarray to
the slide rack in the wash 1, touching the edges or barcode of the slide only.
Part # 15056493 Rev. C
Appendix
Figure 14
Removal of CytoChip Oligo slide from the gasket slide
Equipment List
Table 26 Equipment required for the CytoChip Oligo protocol
Alternative
Recommendations
Company and
Part No.
Name
Description
Benchtop
centrifuge
Variable speed
benchtop centrifuge—
1.5 ml tubes adjustable
from 2000 × g upwards.
eg, Eppendorf
5430/5424
Lidded thermal
cycler
To take microplates
with temperature range
4°C to 95°C.
eg, Applied
Biosystems:Veriti
(4375786)
Swing out
centrifuge
Large centrifuge with
swing out bucket
stainless to hold steel
slide racks. Typically a
microplate swing out
bucket is appropriate.
Agarose gel
electrophoresis
equipment
To enable the
visualization of the
digestion products.
Numerous
options
Spectrophotometer
UV-VIS
spectrophotometer.
NanoDrop ND2000
Evaporator
centrifuge
miVAc Duo (Genevac)
eg, Genevac
#DRS-00000-200
Hotblock
Hotblock to take 1.5 ml
tubes with temperature
range up to 95°C.
Lidded thermal cycler
or 95°C water bath.
eg, Techne
FDB02DD
Agilent
Hybridization
Oven
For hybridizing
CytoChip Oligo arrays
SciGene Mai Tai®
Hybridization System
Agilent (G2545A)
Hybridization
oven rotator for
Agilent Microarray
Hybridization
Chambers
Accessory for fitting
Agilent Hybridization
Chambers into
Hybridization Oven
SciGene Mai Tai®
Hybridization System
Agilent (G253060029)
CytoChip Oligo Microarray Reference Guide
If a swing out
centrifuge is
unavailable, it is
possible to complete
labeling in microplates,
which simplifies
workflow.
eg,
ThermoScientific
Heraeus
Megafuge 40,
Eppendorf 5804
with A-2-DWP
rotor
33
34
Alternative
Recommendations
Company and
Part No.
SciGene Mai Tai®
Hybridization System
Agilent (G2534A)
Name
Description
Agilent Microarray
Hybridization
Chamber
Chambers for clamping
gasket slides to
CytoChip Oligo arrays
400 ml square glass
staining dishes and
lids
To take stainless steel
slide racks.
Part of PR-70431001-00
Clearhyb Wash
system
Precision water bath to
maintain wash
temperature
PR-70-432201-00
(230 V)/PR-70432202-00 (115 V)
Coplin jar
Coverslip removal
eg, Solmedia
SJ320
Magnetic stirrer
Magnetic stirrer with 2.5
cm stir bar.
eg, Bibby Stuart
SB161-3 (3
position stirrer)
Laser scanner
Dual channel
fluorescent laser
scanner with 532 nm
and 635 nm lasers.
Innopsys
Innoscan 710 and
710AL with
Mapix-CS
software
Agilent OzoneBarrier Slide Cover
Kit
Covers the oligo array
surface and protects
against ozone
degradation of signal
Agilent (G250560550)
Part # 15056493 Rev. C
For technical assistance, contact Illumina Technical Support.
Table 27 Illumina General Contact Information
Website
Email
www.illumina.com
techsupport@illumina.com
Table 28 Illumina Customer Support Telephone Numbers
Region
Contact Number
Region
North America
1.800.809.4566
Italy
Australia
1.800.775.688
Netherlands
Austria
0800.296575
New Zealand
Belgium
0800.81102
Norway
Denmark
80882346
Spain
Finland
0800.918363
Sweden
France
0800.911850
Switzerland
Germany
0800.180.8994
United Kingdom
Ireland
1.800.812949
Other countries
Contact Number
800.874909
0800.0223859
0800.451.650
800.16836
900.812168
020790181
0800.563118
0800.917.0041
+44.1799.534000
Safety Data Sheets
Safety data sheets (SDSs) are available on the Illumina website at
support.illumina.com/sds.html.
Product Documentation
Product documentation in PDF is available for download from the Illumina website. Go
to support.illumina.com, select a product, then select Documentation & Literature.
CytoChip Oligo Microarray Reference Guide
Technical Assistance
Technical Assistance
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