Copy Number Variation - How Does It Work? - Ask TaqMan #34

Genotyping single nucleotide polymorphisms, or SNPs, using TaqMan Assays has been very well established. But have you heard about copy number variations?
Copy number variation (CNV) is a type of structural variation that occurs when a DNA segment of 1 kb to several megabases in length is present in variable copy numbers compared to a reference genome. These CNVs can influence gene expression and can be associated with specific phenotypes and diseases. This variation covers approximately 12% of the human genome and includes deletions and duplications. TaqMan Copy Number assays offer a targeted approach combining high accuracy, specificity, ease of use and sample throughput, to validate copy number changes or screen large sample sets.
So how does this all work?
Let’s take a look at our lab book
TaqMan Copy Number assays are a duplex experiment using a FAM labeled target assay and a VIC labeled reference assay in the same well to quantify small fold changes. The reference assay targets a gene of known and stable copy number. For example, RNase P is often used for human samples, as it has been shown to be present and stable at 2 copies.
To determine copy number, a relative quantitation analysis is performed between the unknown sample and a calibrator sample (one in which the copy number of the target gene is known).
The sample is combined with both assays, master mix, and then run in quadruplicate in the real-time PCR instrument.
Let’s look at an example in which the target gene is on the X-chromosome. In a male sample, we should then find a copy number of 1 for the target.
A baseline subtracted cycle threshold or dCt is determined between the two assays. In our example, the unknown sample has a dCt of
1.5 And the calibrator a dCt of 0.5.
We then subtract the dCt of the calibrator from the dCt of the unknown.
Next we convert this value to a relative quantity
And then multiply the relative quantity or RQ by the copy number of the calibrator, which in this case are 2 with RnaseP.
Ah-ha! A copy of 1. Wasn’t that easy? And no messy standard curves to deal with.
Thermo Fisher Scientific offers a free software program which will do all of the analysis for you with the exported data from a compatible instrument. The software provides statistics to help evaluate data confidence and can calculate copy number even when a calibrator sample is not used.
So there you have it! A complete workflow solution for measuring DNA copy number variation in a precise quantitative fashion. Whether you need to quantify copy numbers of important pharmacogenomics genes such as CYP2D6, or you’re investigating copy number variants underlying cancer, Applied Biosystems has the reagents and tools to drive your research.
If you’ve got more qPCR or digital PCR questions, remember, just ask Taqman.
Submit your question at www.thermofishe...

[@ismhj4707]

There is a sign mistake in one of the Delta Cts at 2:06, please correct it. Thank you for the video.

[@CatalinRusnac]

Yup, dCT in the calibrator should be -0.5, but they probably mixed up the Ct(FAM) and Ct(VIC) values

[@nathaliemorales1227]

I want to know where you got that golden pippette! I want one!

[@MonoTheLuke]

Thank you

[@4Getf00l]

How is copy number of a gene analysed? I mean how can I know the copy number of a specific gene?

[@thermofisher]

Thanks for your question Maimuna. When calculating copy number using our assays, there is the use of a reference gene where there is a known copy number.
For example, in humans, RNase P is known to have a copy number of 2 under normal situations. You are using that known number to calculate your unknown target's copy number.
For additional technical support, please reach out to us at thermofisher.com/askaquestion.

[@bientran6391]

how did you do it can you share with me , thank you

[@ellawynn1644]

How would I go about finding specific information due to a 1.6 Mb deletion at 12p.13.32 (3615205-5225595) with at least 18 genes being affected by CNV?

[@thermofisher]

Hi Ella, Thank you for your question. Please contact our technical support team at thermofisher.com/askaquestion for additional information. Thank you!

[@anomalyp8584]

Linus from the genetics department

[@Chicagomom]

How to find a calibrator sample for my gene of interest?

[@thermofisher]

+fan wang Thanks for asking!
Check out this post in the AB Community for information on calibrator samples: abcommunity.thermofisher.com/community/real-time_pcr/blog/2013/08/16/how-to-find-a-calibrator-sample-for-a-copy-number-experiment

[@shixiangwang]

It is too fast.

[@shixiangwang]

But it indeed a good video. Thanks.