I searched several protocols for reverse transcription of RNA and all suggest using RNA concentrations of 1ug. Could a lower amount result in inferior transcript representation and therefore decreased quality of the qPCR results?

We use the SensiFAST SYBR Lo-ROX kit from BIOLINE. I was thinking of using 500ng instead of the suggested 1000ng. In order to accommodate the protocol, I would use 0.5ul of RTase to maintain the 1:1 (m/v) ratio. Buffer, DNase, water, and RTmix concentrations for the reaction I would keep the same. For the final dilution of the cDNA, I would dilute 5x instead of 10x to maintain equal concentrations.

Does anyone have experience with this? Does transcript representation suffer? Do the suggested protocol adaptations seem reasonable?

Thanks for the help in advance.


Two answers follow:

  1. Technical experience of many: it should be no problem to have the same protocol with a twice-lower starting concentration, without making any adjustments whatsoever. The suggested 1ug is very much on the safer side of things, 2-fold fewer transcripts makes almost no difference. Maybe at 1 or 2 orders of magnitude (10 to 100s) less will you run into problems or a need to correct your reactions.

  2. Scientific orthodoxy: the most scientific piece of advice anyone can offer you would be to try it both ways and compare your results using qRT-PCR. I'm guessing both approaches will show very similar results.

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