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Product FAQ

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

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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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 TaqMan™ Gene Expression Assays for a gene copy number study?

Answer

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.

Answer Id: E2736

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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

CopyCaller™ Software gave the error “XXX.txt is not a compatible file and cannot be added to the analysis. Verify that the file format is compatible with the software.” What can I do now?

Answer

Check your export results with the instrument software. The data columns need to be in the following order: Well, Sample Name, Target Name, Task Reporter, Quencher, CT. If needed, rearrange the columns and export the data again for CopyCaller™ Software.

Answer Id: E7559

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Product FAQ

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

Answer

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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Product FAQ

My data will not import into the CopyCaller™ Software. What could be wrong?

Answer

In the instrument software, make sure that you selected “Results” from the export options. The data need to be set up as duplex reactions as well. If you ran the copy number assay and the reference assay in separate wells, the data will not be in the correct format.

Answer Id: E7560

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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

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

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.

Answer Id: E2733

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

Answer

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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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

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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Product FAQ

Which reference assay should I use for copy number analysis with human samples?

Answer

We offer two reference assays for human samples: RNase P and TERT. The RNase P assay is the recommended first choice, with TERT being offered in the event that a chromosomal aberration in a sample results in suboptimal RNase P assay functionality. RNase P is located on chromosome 14, cytoband 14q11.2. The assay location is chr.14:20811565 on NCBI build 37. It has an 87 bp amplicon that maps within the single-exon RPPH1 gene. The TERT assay targets the telomerase reverse transcriptase (TERT) gene located on chromosome 5, cytoband 5p15.33. The assay location is chr.5:1253373 on NCBI build 37. It has an 88 bp amplicon that maps within exon 16 of the TERT gene.

Answer Id: E7549

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Product FAQ

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

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.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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