Literature - Oxford Gene Technology

Literature - Oxford Gene Technology
CytoSure™ Array Handbook
Embryo Screen (8x60k)
CytoSure Genomic DNA Labelling Kit
Oxford Gene Technology — The Molecular Genetics Company™
Founded by Professor Ed Southern, Oxford Gene Technology (OGT) provides world-class
genetics research solutions to leading clinical and academic research institutions.

CytoSure™ arrays — Class-leading products and services offering the complete
array solution for clinical genetics research

Cytocell® FISH probes — High-quality products for the detection of gene
rearrangements related to inherited genetic disease and cancer

SureSeq™ NGS products — Delivering comprehensive, high–quality targeted
sequencing products to clinical and academic researchers.

Genefficiency™ Genomic Services — A tailored microarray and sequencing
service enabling high-throughput, high-quality genomic studies for a variety of
applications
For more information, visit www.ogt.com.
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CytoSure 8x60k Embryo Screen Array Handbook
Contents
Introduction
Pack contents
Storage
Safety
Sample preparation and experimental design
Equipment and reagents required
Protocol: Running OGT arrays
Protocol: Feature extraction
Data analysis
Troubleshooting
Appendix 1: Guidelines for handling single cells
Appendix 2: Minimising Ozone
Legal information
Ordering information
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Introduction
This handbook is for use with CytoSure Embryo Screen 8x60k array using the
PicoPLEX™ WGA kit (Rubicon Genomics) and CytoSure Genomic DNA Labelling Kit. For
information about specific CytoSure products, visit www.ogt.com.
The microarray has been designed to detect amplified material specifically from the
PicoPLEX WGA kit. The use of other amplification methods is not recommended.
Array synthesis
The microarrays are synthesised on the glass using an in-situ ink-jet printer ensuring high
quality microarrays. They are designed to be used in two-colour experiments i.e. one
sample is labelled with one fluorescent dye (Cy3) and another sample is labelled with
another fluorescent dye (Cy5). Usually one sample is the DNA of interest and the second
sample is a reference (or ‘normal’) DNA.
There are eight arrays per slide. It is intended that the arrays are hybridised as
independent hybridisations, so that eight array CGH experiments can be simultaneously
conducted per slide. These hybridisations should be carried out together.
OGT CytoSure microarrays are manufactured by Agilent using SurePrint™ technology and
can be hybridised as Agilent CGH arrays (www.agilent.com). The scanner requirements are
dependent upon microarray format and the feature extraction software. It is recommended
that the 8x60k arrays are scanned at 2 µm.
For further information, consult the CytoSure Interpret Feature Extraction Software User
Guide or contact OGT at products@ogt.com.
CytoSure Interpret Software
CytoSure Interpret Software is a powerful, easy-to-use package for the analysis of aCGH
data, enabling translation of data into meaningful results. CytoSure Interpret Software
offers a large combination of features that allow the choice of standardised data analysis
(using the Accelerate Workflow) or customised, user-defined data analysis. The
functionality allows effortless identification of both copy number variation (CNV) and loss of
heterozygosity (LOH)*. CytoSure Interpret Software is provided exclusively with OGT’s
extensive range of CytoSure aCGH arrays.
For further information, consult the CytoSure Interpret Software User Guide or contact
OGT at products@ogt.com.
* When used in conjunction with arrays containing SNP probes
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CytoSure 8x60k Embryo Screen Array Handbook
Genomic DNA labelling
The CytoSure Genomic DNA Labelling Kit (cat. no. 020020) and CytoSure HT Genomic
DNA Labelling Kit (500040) are optimised for use with oligonucleotide arrays. They use
significantly reduced quantities of dye than alternative labelling kits, yet retain a very high
labelling efficiency and produce good array results with low derivative log ratio spreads
(DLRS). The labelling protocol is fast and straightforward.
This is a low-throughput protocol and the CytoSure DNA Genomic DNA Labelling Kit (cat.
no. 020020) is required.
For further information, contact OGT at products@ogt.com.
Automation products
OGT supplies the Labefficiency™ range of automation products throughout Europe and
recommends SciGene® automated products in the US to streamline the microarray
process. Automation solutions improve test reproducibility and lower costs, while saving
hours of valuable hands-on time. Such solutions provide researchers with a powerful way
to automate the entire microarray workflow, from DNA labelling and purification, through
hybridisation, to array washing and drying. The following systems can be used:
 ArrayPrep® Target Preparation — for automated labelling and hybridisation set-up
 Labefficiency Hyb Oven — for automation compatible hybridisation
 Little Dipper® Microarray Processors — for washing and drying steps
 LabEfficiency O3Zone Workspace and Filter — for protecting fluorescent dyes from
damaging ozone levels, from array washing to scanning
For further information, contact OGT at products@ogt.com.
CytoSure Services
CytoSure Services from OGT — provider of the world's leading high-throughput array
comparative genomic hybridization (aCGH) service — offers a flexible and cost-effective
aCGH processing solution to suit your specific requirements.
CytoSure Services deliver:
 Immediate and cost-effective access to high-resolution aCGH chromosome analysis
 Fast, reproducible and consistent high-quality results, without the costs associated
with setting up aCGH in your laboratory
 The capacity to fulfil your entire current and future constitutional cytogenetic testing
requirements
 Peace of mind through complete sample tracking and expertise at every step of
process
 An industry-leading aCGH analysis package with full complementary training and
support.
For further information, visit www.ogt.com or contact OGT at services@ogt.com.
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Amplify sample DNA
Amplify reference
DNA
Amplified human
genomic DNA
Sample 1
Amplified human
genomic DNA
Sample 2 (reference)
Cy3 labelling
Cy5 labelling
Hybridisation
Wash & scan
Result
Figure 1: Overview of the microarray procedure
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CytoSure 8x60k Embryo Screen Array Handbook
Pack contents
CytoSure Embryo Screen Array (cat. no. 020044)
Component
Contents
Slides
8x60k format oligonucleotide microarray slides
(eight arrays with ~60,000 probes per slide)
CD-ROM
PDF protocol booklets, MSDS for labelling kit reagents, XML and
GAL pattern files for feature extraction, CytoSure Interpret
Software executable file
CytoSure Genomic DNA Labelling Kit (cat. no. 020020)
Component
Contents
Labelling kit
Random primers, reaction buffer, nucleotide mix, Cy3-dCTP,
Cy5-dCTP, Klenow, control λ DNA, water
Storage
The microarrays should be stored in a dehumidified chamber within a light-tight box. The
arrays can be stored for up to 3 months. The CytoSure Genomic DNA Labelling Kit should be
stored at –20C. The Purification Columns should be stored at room temperature (15–25C).
Safety
Take care when handling the microarray slides to prevent them from breaking. If the slides
do break, ensure that protective equipment is used to prevent injury.
Handling of slides should be carried out by trained laboratory staff in accordance with good
laboratory practice, using the correct protective equipment such as laboratory coats, safety
glasses and gloves. Any chemicals used are potentially hazardous. Please refer to the
manufacturer’s MSDS for specific information.
Use of compressed nitrogen should be conducted by trained staff only using the correct
procedures.
Sample preparation and experimental design
Sample preparation
Guidelines about handling single cells are given in Appendix 1.
Experimental design
Two samples labelled with different fluorescent dyes can be hybridized to a single array. Eight
arrays are printed on each slide. Two different experimental designs are possible. The first
option compares test to reference on the same array allowing eight samples to be run. The
section option allows two test samples to be hybridized to the same array. One of the subarrays is used for the reference samples. This approach allows fourteen samples to be run.
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Equipment and reagents required
Labelling of genomic DNA for use on OGT microarrays
Required (not supplied)




PCR block with heated lid
CytoSure Genomic DNA labelling kit (OGT, cat. no. 020020)
Microcentrifuge
PicoPLEX WGA Kit (Rubicon Genomics, cat. no. R300050 or New England BioLabs®,
cat. no. E2620L)
Recommended Equipment (not supplied)

NanoDrop™ spectrophotometer
Hybridisation and washing of OGT arrays
Equipment for the hybridisation of 1” × 3” microarrays
OGT recommends that the Labefficiency system or Agilent SureHyb system is used in
conjunction with the gasket slides. A hybridisation oven with a rotisserie is also
recommended (e.g., Labefficiency — Hyb Oven, cat. nos. oven: 800010; Labefficiency —
Hyb Oven Rotator: 800020, or Agilent, cat. nos.: oven: G2545A; rotisserie: G2530-60029).








Labefficiency — Hyb Oven Chambers (OGT, cat. no. 800030) or SureHyb hybridisation
cassette (Agilent, cat. no. G2534A)
Gasket slides (OGT, cat. no. 500010)
Glass dishes and rack for washing the slides
Magnetic stirrer
A microarray scanner capable of scanning 1” x 3” glass slides. The scanner
requirements are dependent upon microarray format and the feature extraction software.
It is recommended that the 8x60k arrays are scanned at 2 µm.
Dry nitrogen (recommended)
Forceps
Vacuum dessicator (e.g., SpeedVac®)
Recommended reagents (not supplied)



Oligo aCGH Hybridization Kit (OGT, cat. no. 500014)
Human Cot-1 DNA® (Life Technologies, cat. no. 15279-011)
Wash buffers (see table, next page)
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CytoSure 8x60k Embryo Screen Array Handbook
Wash 1
20x SSPE
Volume (1 litre) Wash 2
25 ml
20x SSPE
Volume (1 litre)
5 ml
20% N-lauroylsarcosine
0.25 ml
20% N-lauroylsarcosine
0.25 ml
Water (Milli-Q or similar)
975 ml
Water (Milli-Q or similar)
995 ml
Store wash solutions at room temperature (15–25C).
Alternatively, ready-to-use Agilent Wash 1 (OGT, cat. no. 500015) and Agilent Wash 2
(OGT, cat. no. 500015) may be used.
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Protocol: Running OGT arrays
General procedures







It is highly recommended that powder-free gloves are used throughout this protocol.
OGT arrays should be handled by the edges of the glass slide. The array should not
be touched.
CyDye™ is light sensitive and the labelled target or hybridised slides should not be
exposed to light. Please refer to the CyDye manufacturer’s recommendations.
The arrays are on the same side of the slide as the ‘Agilent’ labelled barcode.
The water used should be molecular biology grade, DNase-free (sterile, 18.2 Ω).
It is recommended that the reference DNA material is extracted from ~10 cells or an
equivalent DNA dilution. As with the sample under analysis, the reference DNA will
require amplification prior to labelling.
If multiple samples are processed simultaneously (>24), please contact OGT as a
high-throughput labelling kit is available.
Amplification of single cell and reference material using the
PicoPLEX WGA kit (Rubicon Genomics or New England
Biolabs)
Important: The reference DNA should be amplified in an identical manner to the sample
DNA. Similar starting amounts of DNA should be used. If the sample used is a single cell
then 50pg DNA should be amplified as a reference.
1. Make up the following master mix:
Volume per sample
Volume per 16 tubes
(8 Cy3 and 8 Cy5)
4.8 µl
84.4 µl*
0.2 µl
3.5 µl*
Extraction Enzyme Dilution
Buffer
Cell Extraction Enzyme
*Master mix contains excess reagents to allow for pipetting error.
2. Add 5 μl to each 5 μl cell sample or control DNA sample.
NOTE: To analyse 14 embryos label one reference with Cy5 and one reference with Cy3,
then 7 embryos with Cy5 and 7 embryos with Cy3.
3. Cycle in a PCR machine as follows:
Temperature ( C)
Time (min)
1 cycle
75
10
1 cycle
95
4
1 cycle
20
hold
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CytoSure 8x60k Embryo Screen Array Handbook
4. Make up the following master mix (Pre-Amp):
Volume per 16 tubes
(8 Cy3 and 8 Cy5)
PicoPLEX Pre-Amp Reaction Mix
84.4 µl
PicoPLEX Pre-Amp Enzyme
3.5 µl
5. Add 5 μl of the Pre-Amp master mix to each tube.
6. Cycle in a PCR machine as follows:
Temperature ( C)
Time (s)
1 cycle
95
120
12 cycles
95
15
15
50
25
40
35
30
65
40
75
40
4
hold
1 cycle
7. Briefly centrifuge and place on ice.
8. Make up the following master mix:
Volume per 16 tubes
(8 Cy3 and 8 Cy5)
PicoPLEX Amplification Reaction Mix
440 µl
PicoPLEX Amplification Enzyme
14 µl
Water
602 µl
9. Add 60 μl of the master mix to the tubes and mix by pipetting.
10. Cycle in a PCR machine as follows:
Temperature (C)
Time (s)
1 cycle
95
120
14 cycles
95
15
65
60
75
60
11. Use immediately or store at –20°C.
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Labelling genomic DNA for use on OGT microarrays (PicoPLEX
amplified DNA)


This protocol requires the use of a PCR block with a heated lid.
Prepare all samples and all references together. Master mixes of reagents reduce
pipetting steps and improve accuracy.
Labelling the target
IMPORTANT: Remove the random primer and reaction buffer from the freezer and defrost
on ice. The dyes and enzymes should be removed from the freezer immediately before
use. Defrost both on ice. Then spin in a centrifuge for 5 to 10 seconds to drive contents off
of walls and lid.
1. Set up the reaction tubes as shown in the table below:
Cy3 samples
Cy5 samples
Master mix (x16)*
Amplified material
8 µl
8 µl
Water
10 µl
10 µl
170 µl
Random primer
10 µl
10 µl
170 µl
Reaction buffer
10 µl
10 µl
170 µl
* Excess reagents have been included
2. Mix by flicking the tubes, then microcentrifuge for 15 s.
3. Denature in a PCR block with heated lid at 95C for 3 min.
4. Immediately place on ice for 5 min.
5. Microcentrifuge for 15 s.
6. On ice, add the following to the tubes:
Cy3 samples
Cy5 samples
dCTP labelling mix
10 µl
10 µl
Cy3-dCTP
1 µl
–
Cy5-dCTP
–
1 µl
1 µl
1 µl
Klenow
7. Mix by flicking the tubes followed by a brief (<10 s) spin in a microcentrifuge.
8. Incubate at 37C for 2 h.
9. Incubate at 65C for 10 min, then place on ice for 5 min.
10. Microcentrifuge for 15 s.
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CytoSure 8x60k Embryo Screen Array Handbook
Purifying the labelled target
1. Prepare the purification columns (supplied in the CytoSure Genomic DNA Labelling
Kit) by vortexing the resin briefly.
2. Loosen the cap one-quarter turn and snap off the bottom closure.
3. Place the column in a collection tube (supplied).
4. Pre-spin the column for 1 min at 2,000 x g.
5. Remove the spin column from the tube and discard the eluate.
6. Place the column in a fresh microcentrifuge tube.
7. Remove the lid from the column, add the sample to the centre of the resin and
centrifuge at 2,000 x g for 1 min. The final volume should be ~45 µl.
8. If a NanoDrop™ spectrophotometer is available, it is recommended that 1.5 µl of
material is used to measure the absorbance at 260 nm, 550 nm and 650 nm. The
amounts of target that should be obtained are shown in the table below.
Approximate amounts
(from a 1.0 µg labelling)
Cy3
Cy5
DNA
6 µg
5 µg
Dye
250 pmol
200 pmol
Note: These are minimum amounts. If the figures are higher, it is still possible to proceed.
9. Combine the labelled targets in one tube.
10. The labelled targets should then be dried to ~16 µl. This may be done using a vacuum
dryer such as a SpeedVac. Dry on low heat in the dark. If over-dried, then resuspend
the pellet in 16 µl of water by gently pipetting up and down.
The labelled targets should be used immediately for hybridisation. If this is not
possible, targets should be stored at –70C in the dark.
Hybridisation of arrays with labelled target
OGT recommends the use of the Labefficiency hybridisation system or Agilent SureHyb
hybridisation equipment. If other hybridisation equipment is used, adjustments to this
protocol may be required.
1. Prepare the 10x Blocking Agent by adding 1,350 µl water to the 10x Blocking Agent
tube (supplied with the Oligo aCGH Hybridisation Kit).
2. Leave at room temperature (15–25C) for 60 min.
IMPORTANT: After use, store the Blocking Agent at –20C for no more than 2 months.
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3. Remove the slide box from its packaging and store slides in a dehumidified chamber.
The slides should be stored in a light-tight box until use.
4. When ready for use, remove the slides from the box. Return unused slides to
dehumidified chamber.
Note: Wear clean powder-free gloves at all times when handling the microarrays.
Handle in a low-dust laboratory.
Note: The arrays are printed on the same side of the slide as the ‘Agilent’-labelled
barcode.
5. Set up the hybridisation. The precise method of hybridisation will depend on the
hybridisation equipment available to the user.
Note: OGT recommends the Labefficiency hybridisation system or Agilent’s SureHyb
equipment, in which each of the eight hybridisations is carried out in a 45 µl volume.
6. Prepare the following in a separate, clean tube. Shake the hybridisation buffer before
use and take extra care when pipetting the viscous hybridisation buffer. The use of a
master mix is recommended as suggested in the table below.
Volumes required for each
45 µl hybridisation mix
Volumes for
master mix (8x)*
Cy3 and Cy5-labelled genomic
DNA
16 µl
–
Cot-1 (1 mg/ml)
2 µl
18 µl
Agilent 10x Blocking Agent
4.5 µl
40.5 µl
Agilent 2x HiRPM
Hybridization Buffer
22.5 µl
202.5 µl
* Excess reagents have been included as the solution is viscous.
7. Add 29 µl of the master mix to each sample and mix thoroughly by pipetting, briefly
spin down in a microfuge for 10 s.
8. Denature the target at 94C for 3 min.
9. Incubate at 37C for 30 min.
10. Quickly spin down in a microcentrifuge for 10 s.
11. Place an Agilent SureHyb Gasket into an Agilent Chamber base or Labefficiency —
Hyb Oven Chambers.
12. Immediately pipette 45 µl of the first hybridisation mix onto the first chamber of the
gasket slide.
13. Dispense the other 7 hybridisation mixes (45 µl each) onto the remaining 7 chambers
of the gasket slide (Figure 2). Record which sample goes in which chamber.
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CytoSure 8x60k Embryo Screen Array Handbook
Figure 2: Gasket slide layout
14. Place an OGT array onto the Gasket slide.
IMPORTANT: Ensure the array is placed with the array side facing down and in
contact with the hybridisation mix.
Note: The arrays are printed on the same side of the slide as the ‘Agilent’-labelled
barcode.
15. If using the Agilent SureHyb system, place the clamp assembly on the slide and
tighten the thumbscrew firmly. If using the Labefficiency assembly, place the cover
over the slides and tighten the thumbscrews firmly.
Some bubbles should form. These bubbles should be moving. If they are not, tap the
chamber on the bench.
16. Place the hybridisation chamber in the hybridisation oven (ideally with a rotisserie).
Hybridise at 65C for 16 h in a light-tight container. Fit the slides vertically and rotate the
chambers at a speed of 20 rpm.
NOTE: Hybridisation can be carried out for a shorter time (eg 6 hours) which will still
allow for whole aneuploidies to be detected. Smaller aberrations may not be detected.
IMPORTANT: Many fluorescent dyes used for microarrays are light sensitive.
Minimise exposure of hybridised microarrays to light. Please refer to the dye
manufacturer’s recommendations.
Washing and scanning of arrays
Before starting, decide if the Agilent stabilisation wash is going to be used (see Appendix 2).
1. Pre-warm Wash 2 and containers to 37C.
2. Ensure that all glass dishes have been cleaned thoroughly with deionised distilled
water. Acetonitrile may also be used. Wear clean powder-free gloves and change
gloves frequently.
Version 3: May 2015 15
3. Place Wash 1 in two glass dishes (one for disassembly and one for first wash).
4. Place the slide in the disassembly bath; gently prise the gasket slide from the
CytoSure array under the surface of the buffer. Transfer the slide quickly to the Wash
1 bath.
Note: The wash solution should be changed every 4 slides.
5. Place a stirring flea in the Wash 1 bath.
6. Stir at room temperature for 5 min.
7. Remove the rack and place quickly into the pre-warmed Wash 2 bath.
8. Stir at 37C for exactly 1 min.
9. Remove the rack, blot and blow dry using air or nitrogen.
10. Scan the slide immediately.
11. Insert the slide into the scanner. Refer to the scanner manufacturer’s instruction
booklet and safety information for instructions about the correct use of the scanner.
Agilent scanner
Insert the slide into the Agilent slide holder, with the array side facing up. The nonbarcoded edge should be placed into the slide holder first. The slide should be scanned
with the green laser (~532 nm) and the red laser (~633 nm). It is recommended to scan at
2 µm resolution. Use of a 20-bit image is recommended. The whole slide should be
scanned.
GenePix® scanner
The slides should be inserted into the scanner array side facing down. The non-barcoded
edge should go into the scanner first. The slide should be scanned with the green laser
(~532 nm) and the red laser (~633 nm). It is recommended to scan at 2 µm resolution. The
whole slide should be scanned.
Visualisation of scanned array images
On printing, each oligonucleotide on an OGT array is assigned a unique feature number.
The identity of the feature number on the array is given the design files which can be found
on the CD-ROM that accompanies the microarrays. As different scanners display the
image of the slides differently, the position of the feature number will vary according to the
scanner.
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CytoSure 8x60k Embryo Screen Array Handbook
Agilent scanner (Agilent image analysis software)
Figure 3 shows how the features are displayed on the screen using an Agilent scanner.
Figure 3: OGT microarray positioned on an Agilent scanner
GenePix scanner
Figure 4 shows how the features are displayed on the screen using the GenePix scanner.
Figure 4: OGT microarray positioned on a GenePix scanner
Protocol: Feature extraction
OGT feature extraction software
Please refer to the CytoSure Interpret Feature Extraction User Guide. This is provided on
the CD-ROM accompanying the microarrays.
Agilent and GenePix feature extraction software
Please refer to the Agilent and GenePix Feature Extraction User Guide that is provided on
the CD-ROM accompanying the microarrays.
Version 3: May 2015 17
Data analysis
CytoSure Interpret Software, supplied with OGT arrays, is a comprehensive array CGH
data analysis and results interpretation package. The software analyses the files
generated following feature extraction and automatically identifies aneuploidies.
The following section explains the process of analysing and reporting data generated from
the Embryo Screen Array.
Load the Agilent or GenePix feature extracted files into the software using the following
method:
File -> Import -> PGS Data and select the files(s) from the file system.
After loading the data, enter the sample IDs, or create new ones if necessary, in the
Assign Sample IDs popup. The data is then automatically analysed using the PGS
settings and the PGS Analysis tab opened for viewing the data.
The PGS Analysis tab
1. Select the Reporting tab.
2. Modify the Report Title and Report Subtitle input fields, this text will be displayed
at the top of both pages of the report.
3. To import a logo select Tools -> Options... -> Report Generation and click the
Change... button in the Logo section. You can then browse to an appropriate file
and select a new image.
4. Complete the fields in the Sample Data section as appropriate.
a. If a required option is not available in a drop-down list, select Other... and
enter the new option, this will be saved for future use.
b. Ensure that the correct Specimen Type is selected as this affects the
automatic interpretation carried out by the software.
c. Options in the Analysed By and Witnessed And Approved By fields
require scanned images of user signatures.
5. If required, enter the details of any reagents used in the processing of the samples.
To add a new reagent to the list, click the Add Reagent button and enter the name
of the new reagent type. This will be saved for future use.
6. Select the Analysis tab. For each sample in the table verify the automatic
interpretation generated by the software. This is done by selecting the correct
interpretation from the chromosome drop-down boxes. Three separate views of the
data are provided to help in making these decisions:
a. Chromosome Segmentation Grid — displays the mean log ratio of the
regions of equal copy number for each chromosome for the selected
sample(s). When an individual chromosome in the grid is selected, a higher
resolution view of the information is displayed in the panel to the right-hand
side of the grid.
b. Chromosome Averages Graph — displays the sample signal intensity of the
probes on each chromosome when compared to that of the reference. All
chromosomes whose values are outside the range indicated by the solid
threshold lines will be identified as either “loss” or “gain” in the table.
c. Genomic View tab — displays the log2 ratio of the sample signal intensity
divided by that of the reference for all probes on the array that map to the
selected chromosome.
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CytoSure 8x60k Embryo Screen Array Handbook
7. Ensure that the Interpretation column is correct. If this is set to Unknown for any
sample, ensure that any “?” chromosome interpretations are modified.
8. To add samples from which no result could be obtained, click the Add “No Result”
Sample button and complete the Sample ID and PB fields.
9. If the selected Specimen Type is First and Second Polar Bodies and there are a
number of missing results, click the Add Missing “No Result” Samples button.
10. To report the X and Y chromosome interpretations separately, ensure that the
Report Gender? check-box is selected.
11. Click the Report button, select a location and name for the document and click
Save. A PDF document will be generated and the software will attempt to
automatically open the file for viewing immediately after creation. In order to be able
to view the document, Adobe Reader must be installed. This can be downloaded
from http://get.adobe.com/reader.
Saving results for future analysis
To be able to view the interpreted data at a later date, click File -> Save As... and select a
location and name for the file in which the information should be stored. This will generate
a .cgh file which can be opened in the software by clicking File -> Open.
Single Channel Analysis
It is possible to analyse up to fourteen embryos on a single slide. The slide must be set-up
as shown below where each letter represents an individual embryo. Each array must
contain one sample labelled with Cy3 and one sample labelled with Cy5.
Ref Cy3 v
Ref Cy5
AvB
CvD
EvF
GvH
IvJ
KvL
MvN
Figure 8: Experimental set-up for single channel analysis
The scanning and feature extraction steps are identical to the dual colour analysis, see
“Visualisation of scanned images” on p19.
Analysing the results
1.
2.
3.
4.
Select Tools and Single channel PGS analysis
Click the Add button to browse to the feature extracted text files
When all text files are loaded click Continue
Select which files contain the reference samples in Cy3 and Cy5, then click
Continue
5. Enter Sample IDs for each reference and click Create Sample. When the IDs have
been entered click Continue
Version 3: May 2015 19
6. Enter the samples IDs for the test samples, taking care to ensure that the correct
dye has been selected and click Create Sample. When all the IDs have been
entered click Continue
7. The data is automatically analysed and can be viewed under the PGS Analysis tab
(see instructions above).
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CytoSure 8x60k Embryo Screen Array Handbook
Troubleshooting
Array hybridisation
Black holes on array
Ensure the correct volume of hybridisation solution has been used.
Take care not to spill any of the hybridisation solution when
setting-up the array.
Check the gasket slide seal has not leaked, if it has report the
failure to support@ogt.com
Fluorescent smears
across the slide
Contamination with fluorescent material during washing. Ensure
clean gloves are worn and forceps are cleaned with water before
and between washes. Any wash containers used should be
cleaned with acetonitrile and copious amounts of deionised,
distilled water.
Contamination from the nitrogen or air used to dry the microarrays
prior to scanning. Change the source of nitrogen or air used.
The arrays have dried out during the hybridisation or wash
procedure. Remove slides from gasket slides under Wash 1.
Carry out a final acetonitrile wash for 1 minute at room
temperature.
Low/no signals on
the array
Poor DNA labelling; check labelling using a NanoDrop. If not
available use a UV spectrophotometer.
Check that the correct side of the array has been scanned.
Check correct side of the array has been used in the array
hybridisation.
Low Cy5 signal
Ensure slides are scanned immediately after washing.
Enclose scanners in a box with ozone scrubbers (such as from
Labefficiency).
Cy5-labelled targets were exposed to light. Cover with foil or use
amber microcentrifuge tubes.
Saturated features
Reduce the PMT setting on the microarray scanner.
High DLRS values
Check the quality of the DNA on a high percentage agarose gel for
degradation. If the DNA is degraded, shown by a smear on the gel,
re-extract the sample.
High background
The most common cause of high background is contaminated
wash dishes. Ensure clean gloves are worn and forceps are
cleaned with water before and between washes. Any wash
containers used should be cleaned with acetonitrile and copious
amounts of deionised, distilled water.
Version 3: May 2015 21
Appendix 1: Guidelines for handling single cells
Protocol for the preparation of single cells, includes polar bodies biopsied from oocytes,
and, cells biopsied from embryos at the cleavage or blastocyst stages.
Reagents required:




Wash buffer (calcium- and magnesium-free PBS containing 0.1% w/v polyvinyl
alcohol)
Rack containing 0.2 ml thin-walled microcentrifuge tubes
Sterile petri dishes
Micropipettes (e.g. stripper pipette) for transferring biopsied cells
The wash buffer can be stored at room temperature or placed in a refrigerator or freezer.
The rack containing the microcentrifuge tubes should be UV irradiated and then kept
sealed and stored in a dust free environment at room temperature.
Cell washing and preparation
Individual cells biopsied from embryos or isolated from other sources should be washed
thoroughly. We recommend that each cell is passed through at least 3 microdrops (5-10 μl
each) of clean washing buffer. If assessing cells from embryos or polar bodies from
oocytes, any cumulus cells still stuck to the zona pellucida should have been carefully
removed before biopsy. This can be achieved by exposure to hyaluronidase. Removal of
cumulus cells reduces the risk of contamination with maternal DNA. The use of
intracytoplasmic sperm injection (ICSI) is essential if PGD is being performed for a single
gene disorder or inherited chromosome rearrangement and may also be advantageous for
cases of pre-implantation genetic screening using microarrays. If the embryos were the
product of regular IVF then please be particularly thorough with the post-biopsy washing
as surplus sperm are likely to be embedded in the zona pellucida surrounding the oocyte
and may introduce paternal DNA contamination.
General notes
The microcentrifuge tubes should be kept closed as much as possible to prevent the entry
of dust. Addition of the biopsied cell should be done in a dust-free, sterile environment,
such as a laminar flow hood. Tubes containing biopsied cells should be kept cool (place
the rack containing the tubes on a frozen gel pack if ice is unavailable), until all cells have
been processed. Wear gloves throughout the procedure and change them if their outer
surface has been in contact with any potential sources of contamination (e.g. skin, dust,
non-sterile equipment, door handles).
Washing the biopsied cells — we recommend depositing several drops of wash buffer
(~10µl each) onto a sterile petri dish. A fresh packet of dishes should be opened for this
purpose and all pipetting should be carried out in a sterile hood.
Once the cell has been transferred to the first wash-drop, the pipette should be washed by
filling with and then expelling wash buffer. This process is repeated three or four times to
clean the pipette. Finally, the pipette is loaded with several microliters of fresh wash buffer
22
CytoSure 8x60k Embryo Screen Array Handbook
and placed alongside the biopsied cell in the first wash drop. To minimise the risk of losing
the cell, it should be observed constantly during washing.
A few microliters of the wash buffer within the pipette are expelled over the cell. This
usually causes the cell to move away from the pipette. Thus, the clean wash buffer
expelled from the pipette washes the cell and pushes it into areas of the drop containing
fresh wash buffer.
The cell is then picked up in a small volume of wash buffer and transferred to the second
wash-drop. Efforts are made to minimise the amount of fluid transferred from one washdrop to the next.
After the cell has been transferred, the remaining fluid within the pipette is discarded and
the pipette is washed twice by filling with fresh wash buffer from an unused wash-drop and
then expelling the fluid. Next, the pipette is charged with a few microliters of fresh wash
buffer and placed in the second wash-drop containing the cell. The cell is then washed as
described above.
This washing process is continued until the biopsied cell has passed through two or three
drops of clean wash buffer. The washing procedure should be thorough enough to ensure
removal of all possible contaminants, but gentle enough to minimise the risk of cell lysis.
Lysed cells often fail to yield results.
Transfer to the microfuge tube — after the final wash is completed, the cell should be
transferred to a microcentrifuge tube in ≤1 μl of clean wash buffer. If the biopsied cell is
transferred in a larger volume of fluid, the reagents used for lysis and DNA amplification
will be diluted to an unacceptable level. The tube should be capped as soon as the cell
has been transferred. Be careful not to expel any air after the cell has left the pipette, as
the resultant bubbles can burst, propelling the cell further up the tube or causing it to lyse.
For microarray analysis, do not overlay the droplet containing the cell with oil.
The tube should be labelled clearly on the lid and on the side and then centrifuged briefly
to deposit the cell at the bottom of the tube. If no microcentrifuge is available in your
laboratory then it is best to pipette the cell directly to the bottom of the tube.
Once a cell has been placed inside a microcentrifuge tube, the tube is sealed and placed
in an empty rack, on ice. Once all of the cells have been processed, they should be frozen
at –80°C or processed immediately.
Version 3: May 2015 23
Appendix 2: Minimising Ozone
Ozone levels in the working environment can be minimised using the Labefficiency —
O3Zone Filter (OGT, cat. no. 800050) and the Labefficiency — O3Zone Workspace (OGT,
cat. no. 800040), both available from OGT. A slide barrier to protect the slide from ozone is
available from Agilent (cat. no. G2505-60550).
24
CytoSure 8x60k Embryo Screen Array Handbook
Legal information
This handbook and its contents are © Oxford Gene Technology (Operations) Limited 2014.
All rights reserved. Reproduction of all or any substantial part of its contents in any form is
prohibited except that individual users may print or save portions of the protocol for their
own personal use. This licence does not permit users to incorporate the material or any
substantial part of it in any other work or publication, whether in hard copy or electronic or
any other form. In particular (but without limitation), no substantial part of the handbook
may be distributed or copied for any commercial purpose.
CytoSure microarrays
This product is provided under an agreement between Agilent Technologies, Inc. and
OGT. The manufacture, use, sale or import of this product may be subject to one or more
of U.S. patents, pending applications, and corresponding international equivalents, owned
by Agilent Technologies, Inc. The purchaser has the non-transferable right to use and
consume the product for RESEARCH USE ONLY AND NOT for DIAGNOSTICS
PROCEDURES. It is not intended for use, and should not be used, for the diagnosis,
prevention, monitoring, treatment or alleviation of any disease or condition, or for the
investigation of any physiological process, in any identifiable human, or for any other
medical purpose.
CytoSure Genomic DNA Labelling Kits
This product is subject to proprietary rights of GE Healthcare Bio-Sciences Corp. (“GE
Healthcare”) and Carnegie Mellon University (“CMU”) and made and sold under license
from GE Healthcare. The Product bearing this label is licensed for sale only for research
(not to include in-vivo research) and in addition may be used to perform research services
for third parties. It is not licensed for diagnostic purposes nor are there any implied
licenses for any other use including, without limitation, commercial uses unless expressly
authorized by GE Healthcare in writing. If you intend to use this product commercially and
you do not have a license to use this product for the intended commercial purpose, return
this product, unopened to Oxford Gene Technology, Begbroke Hill, Woodstock Road,
Begbroke, Oxfordshire, OX5 1PF, UK. and any money paid for the product will be
refunded.
GE Healthcare and/or CMU makes no warranties of any kind, either expressed or implied,
as to any matter including, but not limited to, warranty of fitness for purpose, or
merchantability, exclusivity or results obtained from use of this product, nor shall GE
Healthcare or CMU be liable for direct, indirect, special, or consequential damages such
as loss of profits or inability to use said intellectual property or any applications and
derivation thereof. GE Healthcare and/or CMU does not make any warranty of any kind
with respect to freedom from patent, trademark, or copyright infringement, or theft of trade
secrets and does not assume any liability hereunder for any infringement of any patent,
trademark, or copyright arising from the use of the Product. If you are using this product for
research services, you agree that you will not make any warranty on behalf of GE
Healthcare or CMU, expressed or implied, to any person concerning the application of or
the results to be obtained with the Product.
Version 3: May 2015 25
You hereby agree to defend, indemnify and hold harmless GE Healthcare and CMU, their
affiliates, trustees, officers, employees, attorneys and agents from all claims or demands
made against them (and any related losses, expenses or costs) arising out of or relating to
your and your customers’ use of, disposition of, or conduct regarding the Product including
but not limited to, any claims of product liability, personal injury (including, but not limited
to, death), damage to property or violation of any laws or regulations including, but not
limited to, claims of active or passive negligence.
Patents
The use of the Products is covered by the following patents owned by Oxford Gene
Technology Ltd (a company registered in England under number: 3043815): US
5,700,637, US 7,888,494 and pending applications and the Customer is licensed under
those patents to use the Products only in accordance with these Conditions.
Trademarks
Trademarks: OGT™, CytoSure™, Genefficiency™, Labefficiency™ (Oxford Gene
Technology); ArrayPrep®, Little Dipper®, SciGene® (SciGene Corp.); CyDye™ (GE
Healthcare); GenePix® (Molecular Devices, LLC.); SureHyb™ (Agilent® Technologies
Inc.); NanoDrop™, SpeedVac® (Thermo Fisher Scientific), Human Cot-1 DNA® (Life
Technologies Corp.), PicoPLEX™ (Rubicon Genomics / New England BioLabs®).
Customer’s obligations
The Customer acknowledges that OGT (or a company in the same group of companies as
OGT) owns all intellectual property rights in the design of the Products, including in the
choice and configuration of the oligonucleotide sequences used in the Products and
agrees that:
 it will not disclose more than fifty (50) of the oligonucleotide sequences contained in
the Product in any publication or series of publications; and
 it will include (or ensure that the publisher includes) the following wording in any
publication in which any of the oligonucleotide sequences are disclosed:
“Oxford Gene Technology (Operations) Limited (or its group companies) owns all
intellectual property rights in the design of the microarrays used in this [research]. Such
microarrays may only be reproduced or manufactured by Oxford Gene Technology
(Operations) Limited or with its permission”.
Contact information
Oxford Gene Technology, Begbroke Hill, Woodstock Road, Begbroke, Oxfordshire, OX5
1PF, UK.
Oxford Gene Technology (Operations) Ltd. Registered in England No: 03845432
Begbroke Hill, Woodstock Road, Begbroke, Oxfordshire, OX5 1PF.
Tel: +44(0)1865 856826
(US: 914-467-5285)
Email: products@ogt.com Technical support email: support@ogt.com
Web: www.ogt.com
26
CytoSure 8x60k Embryo Screen Array Handbook
Acknowledgements
We acknowledge the valuable contribution of the lab of Prof. J R Vermeesch at the Center
for Human Genetics, University Hospital Leuven, Belgium.
Version 3: May 2015 27
Ordering information
Product
Contents
Cat. no.
CytoSure Constitutional v3
(8x60k)
Microarray with eight arrays of ~60,000
spots; CytoSure Interpret Software
020045
CytoSure Constitutional v3
(4x180k)
Microarray with four arrays of ~180,000
spots; CytoSure Interpret Software
020046
CytoSure Constitutional v3
+SNP (8x60k)
Microarray with eight arrays of ~60,000
spots; CytoSure Interpret Software
020047
CytoSure ISCA v2 (8x60k)
Microarray with eight arrays of ~60,000
spots; CytoSure Interpret Software
020040
CytoSure ISCA v2 (4x180k)
Microarray with four arrays of ~180,000
spots; CytoSure Interpret Software
020041
CytoSure ISCA v2 (4x44k)
Microarray with four arrays of ~44,000
spots; CytoSure Interpret Software
020042
Genome-wide arrays
CytoSure ISCA UPD (4x180k) Microarray with four arrays of ~180,000
spots; CytoSure Interpret Software
020050
CytoSure ISCA +SNP (8x60k) Microarray with eight arrays of ~60,000
spots; CytoSure Interpret Software
020052
CytoSure ISCA +SNP
(4x180k)
Microarray with four arrays of ~180,000
spots; CytoSure Interpret Software
020051
CytoSure Embryo Screen
(8x60k)
Microarray with eight arrays of ~60,000
spots; CytoSure Interpret Software
020044
CytoSure Chromosome X
(4x44k)
Microarray with four arrays of ~44,000
spots; CytoSure Interpret Software
020015
CytoSure Chromosome X
Array (2x105k)
Microarray with two arrays of ~105,000
spots; CytoSure Interpret Software
020021
CytoSure Medical Research
Exome Array (1x1M)
Microarray with one array of ~1,000,000
spots; CytoSure Interpret Software
020100
CytoSure Molecular Arrays
(8x60k or 4x180k)
Microarray with a choice of formats;
CytoSure Interpret Software. Visit
www.ogt.com to see full list of disorders
covered
Various
Chromosome-specific arrays
Gene-focused arrays
28
CytoSure 8x60k Embryo Screen Array Handbook
CytoSure Custom Molecular
Testing Array (various
formats)
Microarray with a choice of format;
CytoSure Interpret Software
020018
CytoSure Haematological
Cancer +SNP Array (8x60k)
Microarray with eight arrays of ~60,000
spots; CytoSure Interpret Software
020070
CytoSure Cancer +SNP Array
(4x180k)
Microarray with four arrays of ~180,000
spots; CytoSure Interpret Software
700090
CytoSure Consortium Cancer
+SNP Array (4x180k)
Microarray with four arrays of ~180,000
spots; CytoSure Interpret Software
020071
Your choice of array design and format;
CytoSure Interpret Software
020018
Cancer arrays
Custom arrays
CytoSure Custom Designs
Genomic DNA labelling
CytoSure Genomic DNA
Labelling Kit
24 reactions: clean-up columns, dyes,
020020
nucleotide mix, random primers, enzyme,
collection tubes
CytoSure HT Genomic DNA
Labelling Kit
96-Well Plate format for 96 reactions:
96-Well Purification Plate, dyes,
nucleotide mix, random primers, enzyme
500040
CytoSure Interpret Software
Class-leading microarray analysis
software provided with CytoSure arrays.
Email products@ogt.com to request your
demo version
020022
CytoSure Interpret Feature
Extraction Module
Microarray feature extraction module,
compatible with CytoSure Interpret
Software versions 4.4 and above
030022
Outsourced array processing
Enquire
Analysis software
Services
CytoSure Services
For an up-to-date product list and the latest product information, visit www.ogt.com.
Version 3: May 2015 29
Notes
30
CytoSure 8x60k Embryo Screen Array Handbook
Notes
Version 3: May 2015 31
Contact us
Oxford Gene Technology,
Begbroke Hill,
Woodstock Road,
Begbroke,
Oxfordshire
OX5 1PF, UK
T: +44(0)1865 856826; (US: 914-467-5285)
E: products@ogt.com; W: www.ogt.com
Technical support: E: support@ogt.com
T: +44(0)1865 856826
Item number: 990175
32
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