I am working with some very low yield RNA products (single digits ng/uL) and would like to use reverse transcription to generate cDNA for gene analysis by qPCR. The total yield is just over 100ng nucleic acid in about 30uL. I am aware that I could precipitate the RNA again and resuspend in a lesser volume, but each time I precipitate, I cut my yield.
We have the iScript cDNA kit from biorad which we typically use. Does anyone know any techniques or products for boosting my yield of cDNA? The protocol for reverse transcription has a 30 minute reverse transcription step in the thermal cycler followed by a 5 minute rt enzyme deactivation step. Can I simply run this reverse transcription step for longer to increase my yield of cDNA?
It Depends on the cDNA kit, some kits provide cDNA conversion even at less concentration of RNA.
I have also used the High Capacity RNA-to-cDNA kit for low concentration samples, replacing any water in the master mix with additional RNA (so for 20ul reaction use 14.2ul RNA instead of 10ul). You could also increase the amount of cDNA you are putting into your qPCR reactions subsequently. However bear in mind as PCR is exponential you may still be able to detect your target product without problems (10fold decrease in concentration shifts CT by roughly 3.3cycles) - so might be worth testing out what kind of results you get with iscript then qpcr a few test samples first.
The is a kit called 'VILO cDNA kit' specifically designed for situations like this. But, as with all RT enzymes, you'll want to know something about the "RNAse H" activity for each - as that particular function of RT enzymes degrades the RNA as soon as the cDNA is being made - so running for a longer period of time can be counterproductive. Some RT enzymes have been engineered to have a less aggressive RNAse H activity so that cDNA production is 'more highly favored' - but, assuming that your RNA is largely degraded after RT enzyme uses it (as a sense template on which to synthesize anti-sense cDNA) is a safe assumption for the most part.
See:
https://www.thermofisher.com/us/en/home/life-science/pcr/reverse-transcription/cdna-synthesis-kits/rt-real-time-pcr/superscript-vilo-cdna-kit.html
I do not endorse or work for any company - but I have familiarized myself since ~1999 as to what qPCR reagents exist out there in the corporate wild.
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