Top questions from our CNV analysis by digital PCR webinar
Copy number variations (CNVs) are key players introducing both natural genetic diversity and biological dysfunction in humans and are often associated with complex diseases such as cancer, obesity, neurodegenerative and autoimmune diseases. Therefore, the quantitative analyses of CNVs at disease-associated loci provide insights into molecular mechanisms of diseases and offer the potential for the discovery of novel biomarkers.
Digital PCR (dPCR) enables accurate detection of copy numbers in the genome, making it an indispensable tool for diagnosis and subsequent analysis of copy number alterations underlying disease progression.
In this 2020 webinar, R&D scientist Dr. Oezlem Karalay discussed how the QIAcuity Digital PCR System could be used for accurate and high-resolution detection of cancer-related CNVs. She provided insights into experimental optimization and considerations for accurate copy number determination using the QIAcuity Software Suite.
Read this Q&A article summarizing expert answers to the top questions asked during the webinar. You can view the recording here.
Does the CNV analysis mandate the use of one reference assay as a control?
CNV analysis estimates the copy number of target genes in the genome in relation to a known control, a reference assay (or a reference target). Different methods can be selected for normalization of copy number of target genes in QIAcuity Software: A) Reference targets with known copy number/genome can be used for estimation of copy number/genome of target genes in target samples, or B) Reference samples with known copy number/genome of target genes can be used to estimate the copy number/genome of target genes in relation to the reference targets in target samples.
How can we choose QIAcuity Nanoplates for CNV analysis?
The choice of Nanoplate for CNV analysis depends on the desired resolution and the amount/concentration of template available for the testing. The use of 8.5K Nanoplates provides enough resolution for most cases of CNV detection. Depending on the very low DNA concentration of the samples, 26K Nanoplates can be used to increase the total template input amount by loading up to 25 µl into the reaction.
Can digital PCR avoid the use of NGS for CNV assays with the same sensitivity?
Digital PCR can replace NGS for the detection of low frequency known mutations in a high-throughput manner. dPCR, however, unlike NGS, cannot be used to identify unknown mutations. dPCR can be used to validate NGS results for new mutations with high sensitivity and precision.
How much sample can I load for a CNV reaction?
Template loading amounts should lie between 60–250 ng per reaction (up to 7 µl volume) for 8.5K Nanoplate and 75–300 ng per reaction (up to 25 µl volume) for 26K Nanoplate for optimal results when detecting CNV assays that are present in 2 copies/diploid genome.
How many reference assays can I use when setting up a reaction? Does it increase the sensitivity of CNV testing?
We advise using 2 reference assays (or more, if possible) according to the experimental setup. If there is any prior information on expected copy numbers/genome in the test samples, multi-copy reference assays similar to targets' expected copy number/genome should be included in the experimental setup. Increasing the number of reference assays will improve the accuracy of testing.
Can I use any other PCR master mix?
No. dPCR Copy Number Assays are developed for optimal performance using QIAcuity EG PCR MasterMixes. QIAcuity MasterMixes contain a special reference dye required to detect valid partitions analyzed for calculating the copy numbers present in samples.
Is the restriction enzyme provided with the assays?
Enzymes are not provided with the assay kits. However, they are restriction enzymes that are commonly used in the labs. Digestion of the DNA template using restriction enzymes is highly recommended for long DNA templates larger than 20 kb in size or circular DNA to ensure homogenous distribution of the template across partitions. Restriction digestion of templates is critical when multiple copies of the target gene or region of interest might be linked in tandem. Separation of these tandem repeats is required for accurate CNV analysis. Users can set up their dPCR reaction mixes, including restriction enzymes and perform restriction digestion directly in nanoplates. Recommended assays are validated to efficiently digest the DNA templates in 10 min at RT in QIAcuity EG PCR MasterMix without affecting the subsequent PCR amplification of the DNA templates. Recommended enzymes that provide optimal results in QIAcuity EG PCR Mastermixes are listed in QuickStart Protocols. Importantly, restriction enzymes that cut within the target amplicon should not be used. The information about compatible enzymes that do not cut the target amplicon sequence is provided in the product sheet that comes with the kit. It can also be found in the GeneGlobe product specifications.
Can I use pre-digested DNA? If not, how much restriction enzyme do I have to use in a reaction? How much reaction volume do I lose because of the restriction enzyme?
Yes, you can use pre-digested DNA templates. However, we recommend doing digestions in nanoplates for more reliable and consistent sample digestion setups. All samples will be digested equally for the same amount of time, for 10 min at RT. This will also provide more consistent results. Restriction enzymes that do not cut the amplicon should be used in these cases. This information will be provided in the assay product sheets, making it more convenient for the users to select restriction enzymes. We recommend using restriction enzymes at a final concentration of up to 0.25U/µl. This will not affect sample input volumes at all. In 8.5K Nanoplates, up to 7 µl and 26K Nanoplates, up to 25 µl of sample volume can be used.
Do I have to digest FFPE samples?
No. Digestion of the DNA template using restriction enzyme is highly recommended for long DNA templates larger than 20 kb in size or circular DNA to ensure homogenous distribution of the template across partitions. FFPE samples are often highly fragmented and thus do not need restriction digestion.