I need help setting up a reverse transcriptase qPCR assay. I am trying to understand why a few of my RNA samples are failing to produce cDNA products after reverse transcriptase reactions. I want to see if reverse transcriptase qPCR will measure any of my RNA material by converting it to cDNA and measuring the amount of cDNA from the assay.
From my experiment, I have saved aliquots of my stock RNAs that failed, diluted RNA used as input the reverse transcriptase reactions, after mRNA fragmentation (to produce cDNA products of a specific size), and after final cDNA synthesis. I will measure the final cDNA synthesis products using regular qPCR to measure DNA. I have the same aliquots above for control RNA samples that gave cDNA products.
I bought a one step reverse transcriptase qPCR kit and have GAPDH, 28S, and B actin primers. Most people tell me it is the samples but unless I can show that, I can't prove it. I have been able to measure the RNA by RIN score and Qubit and it measures and RIN looks fine. So I don't think it is the samples but something else. I want to see what reverse transcriptase qPCR will show.