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TaqMan CN assay - control sample duplicated

qPCR copynumber CNV

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#1 rjs625

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Posted 18 July 2013 - 04:58 AM

Hi,

We're using TaqMan CopyNumber assays to validate CNVs detected using microarray data. All of our samples that we expect to have CNVs have validated using these probes. However, we are also running additional control samples to rule out CNVs in normal controls. So far we've ran 4 probes and one control sample has been called CN3 (CN4-5 for one probe) for all probes ran. The CNVs we are validating are expected to be rare and not from copy variable regions of the genome. The probes are from different chromosomes, so it is highly unlikely the sample is really amplified in all of these regions. Are you aware of any reason a sample would appear as a CN gain at multiple loci? I've checked the RNase P value for the sample in question and it falls in line with mean of each run, so I don't think an issue with this samples RNase P is causing our results.

I've tried running the qPCR product on a gel and comparing it to other samples and nothing looks abnormal; I see two discrete bands that look comparable to other samples. I've also ran the genomic DNA on a gel and the smear looks similar to samples.

Thanks,
Bob

#2 rjs625

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Posted 24 July 2013 - 04:57 AM

Just an update: I ran this sample using dilutions of gDNA 1:5, 1:10(our usual) and 1:20; all three dilutions were called CN3 is the rerun. I've also ran additional probes on additional chromosomes and they've shown copy number gains (only increasing the odds that these gains are not real). Not sure what else I can do other than omit this control sample.





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