We are doing a RT-qPCR for the first time with Taqman Fast Advanced Master Mix, with the genes GAPDH, BAX, BCL-2, and beta-Actin to run a comparative qPCR. We would like confirmation of how much cDNA we need for the qPCR Taqman reaction, our protocol states 1-100 ng but we were adviced not to measure cDNA after the RT and just use the RNA concentration to calculate the concentration of cDNA.
Thus, if we have 10 ng/μL for our RNA concentration. How much of our RNA do we need to use for our Taqman reaction if we want 15ng of cDNA?
1.5ul.
You assume it's 1:1 conversion. It cannot realistically be _more_, but it can be less, but for all intents and purposes you assume 1:1.
I'd be careful, though: you haven't specified how much you need for each reaction, or whether you want to do multiple reactions, or what. Assuming your RNA is of good quality (which will be hard to tell with such low concentrations), I would recommend using as much RNA as your kit allows, per reaction, because you can always dilute it post-hoc (and usually you should).
Hello Cristina Sanchez Cereceda
I agree with John Hildyard about using 1.5uL of your cDNA to get a concentration of 15ng per reaction. I have used Taqman Fast Advanced Master Mix in the past and from my experience using at least 25-50ng of cDNA per reaction gave me good results. In that case, use 2.5 to 5uL of your cDNA.
Best,
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