General real time questions. - (Apr/30/2010 )
1) DNase of total RNA: I am using Invitrogen DNase 1, Amp grade. I DNase treat 1 ug of total RNA in a final volume of 11 ul. Is it acceptable to reverse transcribe this entire volume to cDNA (~1 ug of RNA--->cDNA)? Subsequently, how much cDNA should be used in my downstream real time PCR if I RT 1 ug of total RNA?
2) -RT controls: I am using Biorad's iScript cDNA synthesis kit. For my no RT control is it acceptable to just add DNased RNA to water to total my final reaction volume or should I be adding the 5X buffer, water and RNA minus the 1 ul of reverse transcriptase?
3) Standard curve: I am looking at a transcription factor and several of it's downstream products in several different tissues (spleen, lung, LN) under control and experimental disease conditions. Will I have to generate a std. curve for each tissue and experimental condition? Obviously I will have to for my GOIs and HKGs.
1) the maximum input amount of RNA should be mentioned in the bio-rad iscript manual but 1µg of RNA is a pretty standard amount and I'm for 99% sure that this will work. i'm mostly using 5 or 10 ng of cDNA (RNA equivalents) in one PCR reaction.
2) do everything exactly like the RT+, just replace the enzyme with water
3) one standard curve for one target is sufficient.