100 ng RNA for cDNA synthesis - (Aug/21/2013 )
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! :)
To both questions, it's fine, go ahead.
I agree with Curtis.
Thank you very much, appreciate it :)
You don't need to normalize. That is the function of the reference gene. What is more important is to ensure you are using the RT kit within the dynamic range, otherwise the cDNA you synthesize may be biased or inhibited. I saw this tutorial the other day; very helpful. www.bio-rad.com/rt_tutorial