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

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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.

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What master mix can I use with the Copy Number assays?

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TaqMan™ Genotyping Master Mix is the recommended master mix for use with TaqMan™ Copy Number Assays. TaqMan™ Gene Expression or TaqMan™ Universal Master Mixes can also be used.

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

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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.

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

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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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What experiment type should I choose in the real-time instrument software for Copy Number Assays?

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In your instrument software, choose “Absolute Quantitation” or “Quantitation - Standard Curve” from the experimental properties. For analysis, we recommend to use an automatic baseline and a manual threshold of 0.2.

Answer Id: E7548

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How much genomic DNA do I need to use with the TaqMan™ Copy Number Assays?

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The recommend input is ~20 ng of genomic DNA. Make sure the gDNA is accurately quantitated, and to use the same amount for each sample run with the same assay.

Answer Id: E7553

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

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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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Can I use the TaqMan™ Copy Number assays for transgenic experiments?

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Yes, we have several assays designed to common reporters used in transgenic studies such as Cre, EGFP, and more. You can find the full list here - https://www.thermofisher.com/order/genome-database/searchResults?searchMode=keyword&CID=&productTypeSelect=cnv&alternateProductTypeSelect=&originalCount=&alternateTargetTypeSelect=&targetTypeSelect=cnv_mr&otherSpecies=&selectedInputType=&keyword=&sequen.

Answer Id: E7543

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What can I do if the copy number confidence values are very low?

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Answer

Confidence values can be low for several reasons. Check your data for the following:

- Large variability in ΔCt values across the plate.
- Low number of replicates per sample (recommendation is for at least 4 replicates per sample).
- Sample copy number is high (> 3). You may want to use copy number bins instead for high copy numbers. Also keep in mind that CopyCaller™ Software is best for copy ranges of 1-5. If you have higher copy numbers, such as with transfections/transductions, it is best to use a standard curve.

Answer Id: E7557

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

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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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What is DGV?

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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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What is a z-score?

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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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Where can I get reference samples for TaqMan™ Copy Number Assays?

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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.

Answer Id: E2733

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

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

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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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