If I do cDNA synthesis with QunatiNova RT kit using 0.5 ug of total RNA in 20 ul RT reaction, would this give better result compared to if I 1 ug of total RNA for the same 20 ul RT reaction?
My worry is, if I use too much RNA in my RT reaction, this might result inconsistent results but at the same time I also want to make sure I have enough cDNA synthesized for qPCR step. I'm aware through literature, both 0.5 ug or 1 ug of RNA to carry our 20 ul RT reaction have been used by other researchers and worked well, but still not sure...
According to QunatiNova RT kit is recommended one can add upto 5 ug RNA template which depends on your RNA quality and integrity (RIN number obtained from Bioanalyzer).
See the mRNA in total RNA is just 3 to %, hence most kits advice to go for 1 to 5 ug of total RNA, let your RIN be between 7 to 9, if you dont have it still you can get good cDNA population, remember just to dilute cDNA 4 to 5 times after synthesis to be used as template for qPCR, mean make up 20 ul cDNA to 100 ul by rnase fee water
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