I have tried to established the amount of RNA input in my RT-qPCRs reactions to amplify interest targets in human monocytes-derived dendritic cells and I perform a RT-qPCR with 4 different RNA amount (6, 3, 1 and 0,75 ng RNA) in 25uL of reaction from 3 different donors, but seems that only in donor A (line A in the plate) got amplification, because in the others donors (line B and C) was detected 2 peaks in melt curve and the highest Tm have the same melt temperature of negative control (withou template), compared to unique peak got in donor A that have higher melt temparature.
Is this the right way to avaliate if I got amplify my interest targets?
The targets was amplified?
Because when I analyze this samples in agarose gel, all RNA amounts of all donors shown the same amplicon
I attached the photos of the analysis to better understand my question
CXCL9 is my interest target and B2M is my endogenous gene
Dear Amanda, this is my opinion:
When u run the gel electrophoresis, u saw the same amplicon for each sample. But when its come to RT-qpcr, only GOI from donor A showed single peak, but not B or C. This becoz RT-qpcr is very sensitive tecnique that can detect a very little amount of unspecific amplification or primer dimer which barely seen in gel electrophoresis. So if your melt curve like B or C, I suggest u increase the annealing temperature and/or reduce the amount of primer. U can try this step when your non-template is ok.
But when I see your non-template result, its similar to donor B and C result. Possibly contaminate? Becoz non-template should not showing any peak.
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