I have total RNA extracted from samples which are rather precious. I am trying to perform cDNA synthesis then qPCR. The RNA appears to be intact and reasonably pure, but I only have between 20 and 100 ng per samples. The reverse transcription kit claims that it can work with as little as 100 pg of RNA. However I have always done qPCR with at least 250 ng RNA, and usually 1000 ng.
Has anyone actually performed RT and qPCR starting with so little material? Would starting with a low amount of RNA potentially skew the results of the eventual qPCR?
Thanks in advance.
Hello, we are performing RT and qPCR for very low amounts of RNA and it works fine. After the extraction, we usually have something like 5 ng/ul RNA. For the cDNA, we use 10 ul of RNA and dilute it up to 110 ul after the RT. And we use 2 ul of that cDNA per each qPCR well. It works fine for us. The RT kit we use is iScript from BioRad and we use SYBR detection.
Variation may increase slightly if you use a lower starting concentration, but using 100 ng should definitely still be fine. I have used 100 and 200 ng starting material with SYBR myself and that works well. I haven't gone lower than that but assume that, just as Nazlı Akın says, it will be ok, definitely if your samples are precious and you can't really make new and better ones.
Hi Stephan Spangenberg
100 ng or less of input total RNA is not considered less. Therefore it should work fine.
I use RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific) to prepare cDNA from 100 ng of input total RNA. In the end, 20 μL of cDNA is prepared for each sample. The cDNA concentration that I get is usually more or less 5-10 ng/μL. This concentration of cDNA works just fine for SYBR-Green realtime PCR. But if you want to use more cDNA, you can just add more μLs of cDNA by decreasing the proportional amount of nuclease free PCR grade water.
Hi Nazlı Akın
Do you know how much primer (ng or uM) were added in the cDNA synthesis step of your reactions?
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