CytoScan™ Dx Assay Kit - FAQs

The tested sources of human gDNA used in the CytoScan Assay are blood and cell line samples.
The genomic DNA (gDNA) sample must be double-stranded, not degraded and free of any contaminants (e.g., PCR inhibitors and other human/non-human gDNA). The recommended starting amount is 250 ng (5 µL with 50 ng/µL) dissolved in low EDTA TE buffer. High EDTA concentration may negatively impact the downstream enzymatic reactions. We recommend running the gDNA samples on a 0.8-1% agarose gel for side-by side comparison with a control DNA (included in the kit). High-quality genomic DNA will run as a major band at approximately 10-20 kb on the gel.
For more detailed information please refer to the CytoScan Assay User Guide, page 9, Genomic DNA general requirements and recommendations section .

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The reagents that require storage at -20 degrees C must not be stored in a frost-free freezer. The activity of the enzymes will be decreased.

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The CytoScan Reagent kit has been validated for less than or equal to 5 freeze/thaw cycles.

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It is critical to have correct size of the fragments for an optimal hybridization. The fragmentation reagent is very viscous and it is easier to get a correct proportion of enzyme when a larger volume is used. The recommendation is to always prepare a master mix for 24 samples even if a smaller number of samples are processed.

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AA markers would be distributed about +1.
AB markers would be distributed about 0.
BB markers would be distributed about -1.

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MAPD: Median of the Absolute values of all Pairwise Differences is a per-microarray estimate of variability, like standard deviation (SD) or interquartile range (IQR). It measures the variability in log2 ratios by looking at the pair difference of all probes and taking a median value. The effect of an occasional big difference in log2 ratios between probes is removed by taking a median value and not a mean. This variability can come from different sources: Intrinsic variability in the starting material, hybridization cocktail preparation, microarray, or scanner.

Apparent variability induced by the fact that the reference may have systematic differences from the sample on this microarray. Regardless of the source of variability, increased variability decreases the quality of the CN calls. A high MAPD can be attributed to any of the above factors and indicates that CN calls may be inaccurate, leading to a higher false positive/negative rate.

SNPQC: This is a measure of how well genotype alleles are resolved in the microarray data. In other words, it estimates the distributions of homozygous AA, heterozygous AB, and homozygous BB alleles and calculates the distance between them. The better the separation of these distributions, the better the ability to identify a genotype based on its cluster position. The larger the difference between the peaks and the troughs, the better the resolution of homozygotes and heterozygotes and the higher the SNPQC metric is. If the three peaks are not well resolved, the difference between peaks and troughs will be low, resulting in a lower SNPQC value. A low SNPQC value indicates that quality of the SNP allele data is compromised, due to higher noise within the array, which compromises the overall quality and clarity of results.

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All genotyping and copy number analysis is done with ChAS. CytoScan Cytogenetics Suite is not intended for genome-wide association studies.

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Yes, it is recommended to run two water shutdown protocols after bleaching the fluidics station.

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Arrays must be put onto the fluidics station immediately after removal from the hybridization oven. Do not remove arrays from the oven you are ready to wash them.

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The hybridization time is 16-18 hrs. If there are a limited number of fluidics stations, the hybridization process can be staggered by two hrs. For example, if there is only one fluidics station and eight arrays, the hybridization for four arrays can be started at one time, and then the hybridization for the other four arrays started two hrs later. After 16 hrs, the first four arrays can be placed on the fluidics station, and then the next four arrays placed on the fluidics station two hrs later. Thus, all eight samples will have hybridized for 16 hrs. Another option is to wash the first set of samples at 16 hrs and the second set at 18 hrs. It is not recommended to stagger more than three wash/stain cycles in an eight-hour workday. Please consult technical support at techsupport@thermofisher.com for more details on the hybridization process.

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The labeled products can be stored at -20 degrees C for no more than 10 days.

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Common causes of under-fragmentation include improper storage or handling of the enzyme, an incorrect volume of enzyme used based on the unit activity, and improper mixing of fragmentation master mix. See the troubleshooting section of the CytoScan Assay User Manual (Cat. No. 703038, https://tools.thermofisher.com/content/sfs/manuals/cytoscan_assay_user_manual.pdf) for more information about under-fragmentation.

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Common causes of over-fragmentation include excess enzyme due to pipetting errors or an incorrect volume of enzyme used based on the unit activity. In addition, warming up of the assembled reaction prior to initiating the 37 degrees C incubation can lead to over-fragmentation. See the troubleshooting section of the CytoScan Assay User Manual (Cat. No. 703038, https://tools.thermofisher.com/content/sfs/manuals/cytoscan_assay_user_manual.pdf) for more information about over-fragmentation.

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No, the routine QC includes running samples only on a gel. Some samples will be saved for additional analysis on the Bioanalyzer, should that become necessary.

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A refrigerated centrifuge is highly recommended. If one is not available, a plastic plate rack that has been stored at -20 degrees C may be used. It is recommended to keep the samples chilled and work quickly prior to initiating the incubation at 37 degrees C.

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Probes on the array are 25-mers. The fragmentation step reduces the purified PCR product size (150- 2,000 bp) to an even smaller size range (25-125 bp), making them more optimal for hybridization to the array probes. Ideally, 25 bp-sized PCR products hybridize to 25-mer probes on the array; however, optimal hybridization can be achieved with 25 to 125 bp-sized PCR products.

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The purified PCR products can be stored at -15 to -25 degrees C for 10 days.

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The purification process is required to remove all the non-amplified DNA after the PCR process.

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The minimum recommended yield is 2.5 µg/µL for a sample. Yields can range from 3.0-4.5 µg/µL, and the average yield for seven or more samples processed in a run (not including the negative control) should be greater than 3.0 µg/µL. If the average yield is below this, consult the troubleshooting section of the CytoScan Assay User Manual (Cat. No. 703038, https://tools.thermofisher.com/content/sfs/manuals/cytoscan_assay_user_manual.pdf).

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Yes, the assay is currently validated for the following magnetic racks:
DynaMag-2 Magnet (Cat. No. 123-21D)
MagnaRack (Cat. No. CS15000)

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No, only the single-tube purification process is validated with the assay.

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A smear in the negative control indicates there has been a contamination. If the negative control band is a light smear, it could be a low-level environmental bacterial contaminant introduced through plastic surfaces. This has shown not to have a material impact on the CytoScan HD data. If a bright band or a smaller smear is seen, then that could be a real cross-contaminant from a sample, and the samples would need to be checked for contamination. If a smear is seen in the negative control well, you should re-run the negative control to verify this was not a result of sample bleed-over from gel loading.

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Using two separate rooms greatly reduces the risk of sample contamination by previously amplified PCR products. If only one room is available, designate one area of the room as the pre-PCR clean area and a separate area as the post-PCR clean area. If a one-room configuration is being used, it is highly recommended to use a laminar flow cabinet for the pre-PCR clean area. See the CytoScan Assay User Manual (Cat. No. 703038, https://tools.thermofisher.com/content/sfs/manuals/cytoscan_assay_user_manual.pdf) for more details about the recommended laboratory setup.

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The digestion step could be root cause. If the reaction did not go to completion, there may be, on average, longer fragments at the end of the reaction. These longer fragments then pass on to ligation and PCR reactions.

Insufficient amounts of PCR primer were added to facilitate suppression PCR. The primer concentration shifts the PCR reaction equilibrium toward larger fragment distributions, in this case, by increasingly unfolding (linearizing) the stem-loop structure at increasing primer concentration when the same adaptor ends hybridize.

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Several things can cause this problem. To troubleshoot this problem, first determine if the positive control worked properly. Common reasons for this failure include incomplete digestion of genomic DNA or inefficient ligation of adaptors, ligation samples that are not properly diluted or mixed, and degraded DNA (if only the positive control worked). See the troubleshooting section of the CytoScan Assay User Manual (Cat. No. 703038) for more information

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A plate can be frozen at -15 to -25 degrees C for up to one week.

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A plate can be held in thermal cycler at 4 degrees C for up to 60 hrs.

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The DNA is digested with Nsp I restriction endonuclease and ligated to adaptors that recognize the cohesive four-base pair (bp) overhangs. All fragments resulting from restriction enzyme digestion, regardless of size, are substrates for adaptor ligation.

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The 10 assay processing steps, as listed in the CytoScan Assay User Manual (Cat. No. 703038) are the following:

1. Genomic DNA Preparation
2. NSP1 Restriction Enzyme Digestion
3. Ligation
4. PCR
5. PCR Product Purification
6. Quantitation
7. Fragmentation
8. Labeling
9. Hybridization
10. Wash, Stain, Scanning

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Always use deionized (DI) water on the fluidics stations for all protocols, including the shut down and bleach protocols.

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Common causes of under-fragmentation include improper storage or handling of the enzyme, an incorrect volume of enzyme used based on the unit activity, and improper mixing of fragmentation master mix. See the troubleshooting section of the CytoScan Assay User Manual (Cat. No. 703038, https://tools.thermofisher.com/content/sfs/manuals/cytoscan_assay_user_manual.pdf) for more information about under-fragmentation.

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Common causes of over-fragmentation include excess enzyme due to pipetting errors or an incorrect volume of enzyme used based on the unit activity. In addition, warming up of the assembled reaction prior to initiating the 37 degrees C incubation can lead to over fragmentation. See the troubleshooting section of the CytoScan Assay User Manual (Cat. No. 703038, https://tools.thermofisher.com/content/sfs/manuals/cytoscan_assay_user_manual.pdf) for more information about over-fragmentation.

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No, the routine QC includes running samples only on a gel. You will save some samples for analysis in case you need them for additional analysis on the Bioanalyzer.

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A refrigerated centrifuge is highly recommended, but if one is not available, you may use a plastic plate rack that has been stored at -20 degrees C. It is recommended that you keep the samples chilled and work quickly prior to initiating the incubation at 37 degrees C. See the CytoScan Assay and Data Analysis Training Video (www.thermofisher.com/us/en/home/life-science/microarray-analysis/microarray-analysis-learning-center/microarray-analysis-training.html) for more details.

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The minimum yield recommended is 2.5 µg/µL. Yields can range from 3.0-4.5 µg/µL and the average yield for seven or more samples processed in a run (not including the negative control) should be greater than 3.0 µg/µL. If the average yield is below this, consult the troubleshooting section of the CytoScan Assay User Manual (Cat. No. 703038, https://tools.thermofisher.com/content/sfs/manuals/cytoscan_assay_user_manual.pdf).

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No, the Magna Rack is the only magnetic rack that is validated with the assay.

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Several things can cause this problem. The first step in troubleshooting this problem is to determine if the positive control worked. Common reasons for this failure include incomplete digestion of genomic DNA or inefficient ligation of adaptors, ligation samples that are not properly diluted or mixed, and degraded DNA (if only the positive control worked). See the troubleshooting section of the CytoScan Assay User Manual (Cat. No. 703038; https://tools.thermofisher.com/content/sfs/manuals/cytoscan_assay_user_manual.pdf) for more information.

Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.

The DNA is digested with Nsp I restriction endonuclease and ligated to adaptors that recognize the cohesive four base pair overhangs. All fragments resulting from restriction enzyme digestion, regardless of size, are substrates for adaptor ligation.

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The CytoScan Assay has significantly fewer pipetting steps and requires less hands-on time than the Affymetrix Genome-Wide Human SNP 6.0 Assay. The CytoScan Assay uses only the Nsp I restriction enzyme and has been optimized for cytogenetics applications. The CytoScan Assay is not intended for genome-wide association studies.

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After any stage in the assay (including fragmentation) you can store the samples at -20 degrees C if you are not proceeding directly to the next step. However, once you have initiated a stage, you must complete it before storage of the samples.

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The expiration date applies to the date of manufacturing. For the CytoScan Reagent Kit, it can be used for up to one year from the date of manufacturing.

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No. The CytoScan Assay has been optimized for performance with the CytoScan Reagent Kit, which is therefore required.

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The CloneTech Titanium Taq PCR Kit (300 reactions, Cat. No. 639240 or 400 reactions, Cat. No. 639243) and absolute ethanol (Sigma Cat. No. 459844) are also required. All other reagents are included in the CytoScan Reagent Kit.

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We recommend running no more than 24 samples at a time. The assay is a four-day protocol; however, the technician can use an optional three-day protocol after becoming comfortable with the assay

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The CytoScan Reagent Kit (Cat. No. 901808) is validated for four freeze/thaw cycles.

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For successful execution of the CytoScan Assay, it is important that your equipment is properly maintained and calibrated per manufacturers' specifications. Please assure that your centrifuges, pipettes, thermal cyclers, and Affymetrix equipment, including the 645 Hybridization Oven, Fluidics Station(s), and Scanner, have had their last recommended maintenance prior to running the assay. For lab equipment, it is typically a yearly calibration. For the Affymetrix equipment, there is typically a yearly preventative maintenance.

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Look at the serial number on the block. An “A” in the serial number indicates the block is aluminum; an “S” indicates it is silver. Also, an aluminum block has a honeycomb appearance between the wells, whereas a silver block is smooth.

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The ABI 9700 (silver or gold-plated silver block only; do not use an aluminum block) and the ABI 2720 (pre-PCR room only) thermal cyclers have been validated for the CytoScan Assay.

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The CytoScan Assay, along with the CytoScan 750K Arrays, the GeneChip Command Console Software (AGCC), and the Affymetrix Chromosome Analysis Suite (ChAS) Software, enable you to perform high-resolution genome-wide DNA copy number analysis. This Affymetrix solution for cytogenetics also provides genotyping information, enabling detection of copy neutral loss of heterozygosity (LOH), which can be used to detect uniparental isodisomy (isodisomic UPD). The combined high-resolution DNA copy number data and the ability to detect gains, losses, and UPDs on a single array makes the CytoScan 750K Solution ideal for next-generation cytogenetics studies.

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MAPD is the recommended method of assessing noise in the SNP Array 6.0, Cytoscan HD and Cytoscan DX copy number data. The log2 distance between neighbors is calculated for all pairs and then the median is taken. This represents the typical distance between marker pairs with respect to log2 ratios. The higher the MAPD, the greater the noise between the array of interest and the reference set.

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