Hey all!
I have a very low concentration of RNA from my samples (1-2ng/uL i.e. a total of 50-100ng). I usually use 500ng of total RNA for my reverse transcription-PCR (reverse transcription) and was wondering if anyone has had any experience (and therefore suggestions) with using a low quantity of total RNA.
Thanks!
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Trof
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Posted 06 December 2012 - 09:33 AM
How did you measured the concentration in the first place? Spectrophotometric methods aren't that sensitive nor accurate.
I would concentrate the samples if they are dissolved in water, and use the whole volume for reverse transcription. You should consult the manual to your reverse transcription kit, what are suitable amounts (for example Roche Transcriptor is suitable from 10 ng of total RNA). But other thing is, if that is enough for detection of your target genes, 10x dilution of RNA may create something like 3 Ct difference from what you measure normally.
I would concentrate the samples if they are dissolved in water, and use the whole volume for reverse transcription. You should consult the manual to your reverse transcription kit, what are suitable amounts (for example Roche Transcriptor is suitable from 10 ng of total RNA). But other thing is, if that is enough for detection of your target genes, 10x dilution of RNA may create something like 3 Ct difference from what you measure normally.
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