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View additional product information for CytoScan™ HD Training Kit - FAQs (901834)
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Here is the link to the CytoScan assay user guide: https://assets.thermofisher.com/TFS-Assets/LSG/manuals/703038_cytoscan_assay_UG.pdf
In Appendix D (starting on page 67), the manual outlines specifications for compatible thermocyclers. Any thermocycler that fits those specifications will work.
Note: Use the silver or gold-plated block only, do not use an aluminum block. In order to determine which block you have, look at the serial number on the block. An "A" in the serial number indicates that the block is aluminum; an "S" indicates that it is silver. Also, an aluminum block has a honeycomb appearance between the wells, whereas a silver block is smooth.
Methods that include boiling or strong denaturants are not acceptable, because the DNA would be rendered single-stranded. For genomic DNA extraction, we recommend using either the Qiagen Gentra Puregene Kit or the 5 Prime PerfectPure DNA Blood Kit.
Our CytoScan HD Assay requires genomic DNA concentration ≥50 ng/µL. Therefore, the elution volumes for each of the kits will need to be adjusted accordingly to achieve the desired concentration.
The presence of RNA and free nucleotides can interfere with some quantitation methods using a spectrophotometer or a NanoDrop instrument. To eliminate RNA contamination, perform RNase treatment during extraction as follows:
- For QIAGEN Gentra Puregene Kit: Perform RNase treatment as recommended in the extraction kit manual prior to elution of genomic DNA.
- For 5 PRIME PerfectPure DNA Blood Kit: Use only RNase-treated purification columns for extraction of genomic DNA.
The purified genomic DNA extracted using the two methods above should meet the DNA quality specifications per the manufacturer’s kit extraction manual.
Note: Both blood and cell line sample sources have been tested for the CytoScan HD assay.
The CytoScan HD Array includes 750,000 "genotype-able" SNPs and 1.9 million non-polymorphic probes. The CytoScan HD Array covers both constitutional and cancer genes with:
- ISCA constitutional coverage at 1 marker /384 bases
- Complete cancer gene coverage at 1 marker / 553 bases
- 12,000 OMIM genes at 1 marker / 659 bases
- >36,000 RefSeq genes with 1 marker / 880 bases
- Backbone (non-gene) coverage of 1 marker / 1,737 bases across genome for breakpoints
Array format: 49
Feature size: 5 µM
Time to scan per array: ~32 mins
Hyb temperature: 50 degrees C
The CytoScan HD Array uses the following QC metrics:
- SNPQC ≥15
- MAPD ≤0.25
- Waviness SD ≤0.12
The Purification Wash Buffer and Elution Buffer are not affected by being frozen at -20 degrees C and are safe to use for purification.
A subset of Y probes is used and run through a Hidden Markov Model to determine gender. Copy number of 0 or 1. 0 indicates female and 1 indicates male.
Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.
The Minor Allele Frequency (MAF) refers to the frequency at which the least common allele occurs in a given population.
For example: A SNP with an allele (G) frequency of 0.40 implies that 40% of the population has G allele versus the most common allele (major allele), which is found in 60% of the population. If an SNP allele frequency is 0.05 in a population of 100 people, the number of alleles for each SNP is: 100 * 2 = 200 (each individual has two alleles).
Total number alleles for your SNP with a MAF of 0.05: 200 *0.05 = 10.
The minor allele is present ten times in your population. However, you don't know how many are heterozygotes vs homozygotes.
Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.
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. Therefore, our recommendation is to always prepare a master mix for 24 samples, even if a smaller number of samples are processed.
Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.
No, we do not offer probes in the Cytoscan HD array that cover the mitochondrial genome.
Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.
There are 549,673 SNPs that are genotyped on CytoScanHD which match to a marker on the Genome-Wide Human SNP Array 6.0.
Note: CytoScan HD Array can be ordered as part of the following kits: CytoScan HD Array Kit and Reagent Kit Bundle (Cat. No. 901835) and CytoScan HD Training Kit (Cat. No. 901834)
Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.
We recommend using the following thermal cyclers with Cytoscan HD:
For pre-PCR, we recommend 2720 Thermal Cycler (Cat. No. 4359659)
For pre- and post-PCR , we recommend the following thermal cyclers:
- GeneAmp PCR System 9700 Gold (Cat. No. 4314878) (Note: This instrument has been discontinued and therefore cannot be ordered)
- GeneAmp PCR System 9700 Silver (Cat. No. N8050001) (Note: This instrument has been discontinued and therefore cannot be ordered)
- Applied Biosystems Veriti Dx 96-Well Thermal Cycler (Cat. No. 4452300)
- Applied Biosystems Veriti 96-Well Thermal Cycler (Cat. No. 4375786)
- Bio-Rad® DNA Engine® PTC-200 Thermal Cycler
- Eppendorf® Mastercycler® Pro S Thermal Cycler
Note: You must use thermal cyclers with silver or gold-plated silver blocks. Do not use thermal cyclers with aluminum blocks.
Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.
Tough-Spots® labels are small adhesive stickers used to temporarily seal the backs of cartridge arrays during the overnight hybridization step. They are required to prevent loss of volume due to evaporation through the septa. We recommend using Tough-Spots® labels on Rolls from USA Scientific (Item No. 9185-0000)
Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.
Proper hybridization volume is critical to obtaining an even signal across a given array. Too little volume can lead to black circles in the middle of the array. Too much volume can leak out of the back of the array. The correct hybridization volume leaves enough room for a small air bubble to circulate around the array surface during the overnight hybridization. Here are the recommended hybridization and fill volumes based on the array format:
Array Format; Hybridization Volume; Fill Volume
- 49 Format (Standard); 200 µL; 250 µL
- 64 Format; 200 µL; 250 µL
- 100 Format (Midi); 130 µL; 160 µL
- 169 Format (Mini); 80 µL; 100 µL
- 400 Format (Micro); 80 µL; 100µL
Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.
The hybridization temperature is 50 degrees C. The rotation speed is 60 rpm.
Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.
Cytoscan HD arrays use the following QC metrics: SNPQC≥15, MAPD≤0.25, Waviness-SD≤0.12.
Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.
| Applied Biosystems Veriti 96-well | Bio-Rad DNA Engine PTC-200 | Eppendorf Mastercycler Pro S | |
|---|---|---|---|
| Lid temperature | 103 degrees C | 103 degrees C | 103 degrees C |
| Temperature mode | N/A | Calculated | Safe |
| TSP heated lid | N/A | N/A | Yes |
| Switch off lid at a low block temperature | N/A | N/A | No |
The post-PCR lab contains amplicons, whereas the pre-PCR area should not. These amplicons can be inadvertently transferred back to the pre-PCR area, thus contaminating the lab and posing a contamination threat to all reactions set up in the pre-PCR lab. Because all samples are amplified using the same primers and adaptors, contaminating DNA can also be amplified.
Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.
Positive control: This sample has prior proven performance and represents a pristine sample, meeting all genomic DNA input requirements. The performance of this sample helps to assess the quality of the assay steps and final product. In troubleshooting cases, performance of this control sample versus that of non-control samples can help to parse out possible assay failure culprits.
Negative control: For this assay, the negative control is simply reduced EDTA TE Buffer and acts as a control for the process up to the PCR quantitation and PCR gel. If no smear is seen on the PCR gel, it is considered passing, indicating that there is no DNA contamination in any of the reagents that could lead to amplification of other targets. A failing result will have a smear in the PCR gel. Note that the presence of PCR primer dimers is expected.
Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.
No, the reagents and arrays are sold as bundles for 24 reactions (Cat. No. 901835)
Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.
The CytoScan assay has significantly fewer pipetting steps and requires less hands-on time than the 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.
Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.
The expiration date applies to the date of manufacturing. For CytoScan Reagent Kit, the reagents can be used for up to one year from the date of manufacturing.
Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.
No. The CytoScan assay has been optimized for performance with only CytoScan Reagent Kit.
Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.
It is highly recommended not to mix different sub kit lots from different modules because functional testing is done based on sub kits from the same lot.
Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.
Only the volume that is indicated on the label can be guaranteed. Since our assurance guarantee is only provided for the label volume, it is advised not to plan experiments or standard operating procedures based on potential overage volumes.
Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.
It is recommended to run 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.
Find additional tips, troubleshooting help, and resources within our Microarray Analysis Support Center.