I performed a qPCR for one gene in THP1 cell line cDNA (32 samples and 6 standards). The results shows 3 samples and one standard peak at different Tm in melt curve. What could be the reason?
Hi Virendra,
Have you tried checking the specificity of your gene primers using a tool such as PRIMER-BLAST? I have found in my experience of qPCR in cell lines that sequence similarity and variant forms of the same gene sequence can be found. Preparation of the cell lines (RNA extraction/cDNA transcription) is sometimes a culprit for variations observed. It may be possible then that different off-target amplification products are also present.
Also have you tried checking this on a gel electrophoresis and that one clear band is created at the different Tms seen?
Thanq Valerie for you prompt reply....
Yes i used primer blast tool, as well i checked in gradient PCR followed by gel for specific single band.... well other genes are working fine..i found such issue first time.... only in three samples n a std. i had problem..rest all ok...their peak shifted to little lower temp
A picture would be informative to obtain a profile of their potential size.
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