I have synthesised cDNA from 500ng/ul of RNA. The concentration of the cDNA Is about 5000ng/ul. Should I dilute my cDNA or just use it as it is for my qPCR? I usually use 2ul of cDNA for a 20ul reaction. Thank you!
Optimal performance of qPCR shall be in the range of 1-100ng cDNA/ 20µL reaction mix. But it really varies according to the gene of interest (basal gene expression, for example). I would recommend dilution. If you want to be more accurate, do a serial dilution, test them by qPCR.
How are you calculating the concentration of your cDNA? Reverse transcription should be (ideally) 1:1, so if you start with 1000ng of RNA (total), you should assume you have ~1000ng cDNA (total).
Once you have made cDNA, do not spec it (nanodrop etc), because cDNA reactions will still contain all the oligodT, random primers, free nucleotides etc that were part of the synthesis reaction: all these will absorb at 260, giving you a completely misleading value for [cDNA]. Assume 1:1, proceed from there.
In terms of "amount of cDNA per well", by your calculations you're using 10000ng: even if we account for the possibility your [cDNA] calculations are way off, this is far, far too much. I use ~8ng, even for low abundance transcripts. 8ng of cDNA is a _ton_ of template molecules.
So, basic overview: determine how much RNA to use (most kits are 800-2000ng per reaction), determine what volume of RNA that is. Make cDNA, assume 1:1 conversion (so if you had 800ng RNA, you now have 800ng cDNA, probably in a 10-20ul reaction). Dilute cDNA at least 1/10 in nuclease free water (I usually use 1/20) to dilute out cDNA synthesis components. Use 1-2ul of this diluted stock per well (giving ~4-8ng of cDNA).
Generally I would do a 1:100 dilution of cDNA for qPCR. I find that this will generally get you in the correct range.
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