How much oligo dT cDNA to use in PCR - (Oct/31/2008 )

Hi all,

I was wondering if anyone could give me a rough estimate of how much cDNA (in terms of nanograms) generated using oligo dT primers should be used in a standard 25 ul PCR reaction. My gene of interest is "relatively low copy" but I don't really know anything more than that.

Thanks,
Kevin

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This is hard to tell. Usually when you use an inactivated RT reaction, the maximum volume recomended is 10% of PCR reaction (due to the possible inhibiting effect of the RT mix). And the endpoint concentration depends on how much RNA you put there first, since cDNA RNA/DNA hybrids after RT are AFAIK not measurable on spectrophotometers.
For real-time we usually use 1ug of RNA for a 20ul RT and then take 2 ul of the RT reaction and the low copy genes are detectable.

So if you already have a unpurified cDNA, use maximum (i.e 2.5 ul) of the RT and see if you get your gene. If not, put more RNA into RT.

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Thanks Trof!

Are you doing 25 ul real-time PCR reactions?

I use RNAse after cDNA synthesis. I use a NanoDrop to quantify my RNA but I have read that unpurified first-strand cDNA can't really be measured accurately because of all the RT reaction leftovers. Do you agree with that?

I will take your advice and give that a try.

Thanks again,
Kevin

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We use 25 or 20 ul reactions, yes, I forgot to specify.

We never tried to measure cDNA quantity, the reason is probably the one you posted. To test the quality of cDNA we do a rutine beta-actin PCR, it's very abundant in every cell. And estimate, the RT eficiency is more or less the same, when using the same protocol.

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