Hi,

During QPCR, I ran non template controls and negative control RT reaction (no RT enzyme using random hexomers and oligodT primers) together with my normal cDNA samples in the same plate. After qPCR, I ran the PCR products on a gel to identify the bands for the product size. Note that all my primers are exon spanning.

The problem is that -RT samples showed amplification with expected size bands. The ct values were higher for -RT’s but not high enough to be negligible especially. If the ct value for my normal cDNA is 18, the Ct value for my -RT is 24. The reason for this cannot be genomic DNA contamination as the size of the bands are the same as the bands I got for the normal cDNA samples.

I still increased DNAse incubation time to account any effect of the gDNA but the ct values only increased by 2-3 values which still was not negligible.

I also run non template controls which did not show any ct values nor a band when I ran a gel. In this case I don’t think I have a contamination problem either.

When I ran the RNA only for some of them, I still had amplification with ct values similar to -RT controls.

I am worried that DNA polymerase is amplifying the RNA that is left in -RT samples. Do you have any other explanation for this? What can I do to prevent amplification in -RT samples? Any other thoughts?

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