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Product FAQ

I am working with a species other than human and mouse. What reference assay can I use for copy number analysis?

Answer

First, you would need to select a reference sequence: usually a well studied gene, known to be present in 2 copies per diploid and found in a non-copy-number variant region of the genome. Next you can use Primer Express™ software to generate a VIC™/TAMRA™ probe-based assay from the sequence. The primers and probe sequences can be ordered online. A 20X stock reference assay should be made that contains 18 μM each primer and 5 μM probe. The reference assay should be tested on a number of samples (4 replicates each) to determine its performance before using it in duplex with CN assays. [Note: Primer Express™ Software comes free with any Applied Biosystems™ real-time PCR instrument].

Answer Id: E7550

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Product FAQ

What should I do when I get the CopyCaller™ Software error “Insufficient data points to estimate confidence and z-score values”?

Answer

This message will appear if you don’t have at least 7 samples in a given copy number group. You can continue with the analysis, but just be aware that you will not get any statistics with your data.

Answer Id: E7554

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Product FAQ

What is a z-score?

Answer

The |z-score|(absolute z-score) is a statistical measurement that indicates the number of standard deviations an observation is above or below the mean of a normally distributed population. The z-score is examined when the confidence call for a sample copy number is high, e.g., above 95%. When the confidence is below 95%, it is not necessary to review the |z-score|. The |z-score| shows how meaningful the copy number call is; i.e., shows if the sample deviates significantly from the copy number mean for other samples having the same copy number call. The following guidelines below are recommended for sample copy number calls having a confidence value > 95%, if the |z-score| is: <1.75, trust the copy number call (pass); between 1.75 and 2.65, the call is borderline (pass with caution); >2.75, do not trust the call (fail).

Answer Id: E7539

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Product FAQ

Can I run the TaqMan™ Copy Number Assays in singleplex?

Answer

The TaqMan™ Copy Number Assays must be run in duplex for good quantitation results. Inclusion of the reference assay in the same reaction as the copy number assay normalizes for the amount of DNA in that particular replicate well. The CopyCaller™ Software cannot analyze data from a plate running singleplex assays.

Answer Id: E7551

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Product FAQ

When trying to open CopyCaller™ Software, the program freezes after launch and will not open. What can I do?

Answer

In this case, you may see the blue CopyCaller™ Software screen pop up, but it will never advance beyond this screen to actually open the software. If this occurs, try the following:

- Press Control+Alt+Delete to bring up the Task Manager.
- Select the “Processes” tab, and look for a process called “javaw.exe”. End this process and then relaunch the CopyCaller™ Software.

Answer Id: E7555

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Product FAQ

What is a calibrator sample? Is it necessary for my experiment?

Answer

A calibrator sample is one in which the copy number of the target gene is known. It is helpful to have for analysis, but if you do not have one the CopyCaller™ Software can still do the analysis. If a calibrator sample is not present, the software will apply an algorithm that calculates copy number assignments that maximizes the likelihood of the observed data with respect to a theoretical model.

Answer Id: E7540

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Product FAQ

How do I find a calibrator sample for my qPCR experiment? Is it necessary for my experiment?

Answer

We recommend that you check the DGV (database of genomic variants) database for your CNV of interest. Not all regions of variance have control samples available, but if there are controls for your CNV, the DGV will list some Coriell sample IDs. You can then order these control samples from Coriell. You can check out our blog post (http://lifetech-external.hosted.jivesoftware.com/community/real-time_pcr/blog/2013/08/16/how-to-find-a-calibrator-sample-for-a-copy-number-experiment) on how to find a calibrator sample for CNV experiments for some screenshots on what to look for in DGV.

Answer Id: E7544

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Product FAQ

Can the TaqMan™ Copy Number Assays be run using a Fast protocol?

Answer

The TaqMan™ Copy Number Assays have not been validated with a Fast protocol. Preliminary studies have shown that Fast protocol analysis is not reliable for copy number quantitation analysis.

Answer Id: E7552

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Product FAQ

What TaqMan™ Copy Number Assays are available now?

Answer

We have predesigned TaqMan™ Copy number assays for human and mouse, and we also have the Geneassist Copy number assay workflow builder tool that you may use to submit sequences for custom designed assays. Copy number variation experiments require a reference assay run in duplex with the target assay. We offer RNase P and TERT reference assays for human, and we offer Tfrc and TERT reference assays for mouse. The copy number assays are FAM-MGB labeled and the copy number reference assays are VIC-TAMRA labeled.

Answer Id: E2728

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Product FAQ

What can I do if the reference assay Ct values vary during copy number analysis?

Answer

Check your pipettes for accuracy. Check the concentration of your samples and make sure they are normalized. Another possibility is that the chromosome on which the reference gene is located is disrupted in the samples you are working with. In this case, try a different reference assay.

Answer Id: E7556

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Product FAQ

Can I use the CopyCaller™ Software if I am using a non-Applied Biosystems™ Real-Time PCR instrument?

Answer

Yes, although the data will not be directly compatible. You can use the example files that install with the CopyCaller™ Software as a template to use with your data. You will have to copy/paste in the sample/target names and Ct values before importing into CopyCaller™ Software.

Answer Id: E7561

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Product FAQ

What is DGV?

Answer

DGV stands for Database of Genomic Variance, which is an online database of human genomic structural variation information. When applicable, every TaqMan™ Copy Number Assay will list the DGV variant ID that the assay is target, and links out to the database.

Answer Id: E7541

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Product FAQ

My results are different from what I expected when working with your Copy Number Variation Assay. Is there something wrong with the assay?

Answer

Check that the reference assay is performing consistently across all samples. Check the concentration of your samples and make sure they are normalized. We also recommend you to use several copy number assays targeting the same variation to validate the data.

Answer Id: E7558

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Product FAQ

Can I use the TaqMan™ Copy Number assays to determine high-copy insertions?

Answer

Our copy number analysis by ddCT is best for up to 5 copies. This is because as you get to higher copy numbers, the fold change between copies gets smaller and smaller. For copy numbers above 5, it is best to use another method such as digital PCR or standard curve analysis.

Answer Id: E7545

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Product FAQ

Do I need to do a validation test before I use the ΔΔCt method to determine the copy number of the target gene?

Answer

If you are using our copy number assays and reference assays, this is not necessary.

Answer Id: E2734

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