I would like to know when running a no rt control for qPCR should the amount of template used be the same as the amount used to make cDNA or the amount of cDNA used in the qPCR. I use 100ng of RNA to make cDNA but run 1ug amounts of this in my qPCR.
When you setup this control, you want to avoid changing anything except the omission of the RT. Best practice would be for you to carryout the cDNA reaction without RT, then use that for your qPCR, exactly as you do for your other qPCR rxns. Good luck.
Ideally, for no RT control, you should be setting such reaction while doing cDNA synthesis by including all components in the reaction mixture but omitting the reverse transcriptase enzyme when doing a cDNA synthesis in the 1st step. From this you can use sample volume of reaction mix for 2nd step as that of cDNA.
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