I hv a question, it maybe sound silly for you but I want to make sure. I extracted RNA from frozen tissues, and some give a 300-400 ng/ul concentration, but some give a very low amount RNA (15-20 ng/ul) concentrations. Since I have to do real-time PCR for these samples, I gotta normalize the RNA samples to the same concentration for cDNA synthesis. From the nanodrop result, 500 ng total RNA for cDNA synthesis is not possible for the low-amount-RNA samples (the final volume for cDNA synthesis is 20 ul), so I want to normalize all samples to 100 ng. So that's what I wanna ask, is 100 ng total RNA for cDNA synthesis considered as too low or not??
PS: I already have synthetized some samples using 100 ng total RNA and it went well (the qPCR can detect it) but I'm afraid I use too much component for it. E.g: the cDNA synthesis protocol said: use the final conc. 60 uM random hexamer primer (equal to 2 ul) for 1 ug total RNA. Can I use the same amount for my 100 ng total RNA samples?
Oh, and I also re-run the RNA extraction for the low-amount-RNA samples but nothing change much. The concentrations are low too.
Thank you, any help will be appreciated!