What TaqMan® Copy Number Assays are available now?

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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.

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e1ec71c187ca4819a579c381d20c9fe9_FAQ

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

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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.

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5000fdec292ec23d000289401d0ce21d_FAQ

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

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Answer

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

Answer Id: E2734

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708c9fb44c4138ab0fcbbd5125eee6cc_FAQ

What type of samples should be run with TaqMan® Copy Number Assays?

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These assays target regions of variance that are found in genomic DNA.

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c7b4a5b26fcf1188a7799ea8a8577c0d_FAQ

Can I use TaqMan® Gene Expression Assays for a gene copy number study?

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TaqMan® Gene Expression Assays with “_s1” as assay ID suffix are the assays whose primers and probes are designed within a single exon, and therefore, will detect genomic DNA. However, the application of these assays in gene copy number studies has not been validated. Other gene expression assays will not detect gDNA, let alone the region of variance.

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9b88975d86be5c1cd3d0c7c76b1ac8c2_FAQ

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).

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feaba64e7524090bf54d13ccc482b922_FAQ

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

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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.

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ec13a0577b05fba9309ea2d1e1ecff53_FAQ

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.

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8fef8ce6a2803a84cbb01d57a52c9be8_FAQ

How do I analyze my copy number data?

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Answer

We recommend you to use our free CopyCaller™ Software. The CopyCaller™ Software was developed specifically for TaqMan® Copy Number Assay data analysis. This free, easy-to use software utilizes a graphical interface and quickly calculates the possible copy numbers for a set of samples in a run. It also estimates a confidence value for each copy number call and has outlier removal functionality.

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acb6840b38462caf5763d7a147e6c5c8_FAQ

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

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Answer

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.lifetechnologies.com/order/genome-database/searchResults?searchMode=keyword&CID=&productTypeSelect=cnv&alternateProductTypeSelect=&originalCount=&alternateTargetTypeSelect=&targetTypeSelect=cnv_mr&otherSpecies=&selectedInputType=&keyword=&sequen.

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f3896a750d115b0e2ea6fe90d31d589d_FAQ

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

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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.

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1ebe6d78e8629da6781d80b378092c4c_FAQ

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

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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.

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7bc9c7f1989f79e3853eabe72ba2d77d_FAQ

How many replicates should I run in my TaqMan® Copy Number Assay?

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We recommend that you to run 4 replicates per sample for reliable copy number calls.

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88e3268e4354f09ac93bc57eab0a8e33_FAQ

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

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Answer

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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dc3d22d0d1810060fdb2cee4c1bedfcc_FAQ

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.

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