Hi everyone, I am trying to do qPCR on mouse gastrocnemius muscle. I am using Qiagen kit for muscle RNA extraction and I do not see any genomic DNA band in my RNA gel. However, when I run qPCR for GAPDH and HPRT1 gene primers (tried more than 1 primer set), I get a peak on my sample cDNA as well as NRT (no-reverse transcriptase ctrl) cDNA from the same sample. No peak for NTC (no template control) cDNA of same sample. This peak in NRT ctrl is almost 5-10 Ct apart from my sample cDNA peak but at the same size on the gel.
Does anyone has any insights what could be going on here? I will really appreciate all the inputs.
Hi Nipun, it looks that your primers are amplifying genomic DNA. Ty to design intron-spanning primers or design within two adjacent exons and / or use DNAse removal treatment and if you are certain that gDNA is not the culprit here so one possibility is that often improperly designed primers have enhanced self-complementarity so they can amplify together and give proper amplification signals. It has also reported that if any primer spans two exons of a gene within which a smaller intron exists so gDNA may get amplified during qPCR, but your gel picture would be negative for genomic the DNA. So work more on primer designing. I hope it will solve this issue.
Regards....
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