Use PCR products to determine efficiency of amplification? - (Apr/19/2006 )

Hello all,

I am wondering if anyone can tell me whether it is possible to use purified PCR products to determine the standard curve, in order to work out amplification efficiency?

I have used PCR products for optimisation and they seem to work well.

thanks

sure, why not?

just be certain to purify and quantify very cafefully and I don't see any reason why that's not OK

Thanks for that Aimikins,

I'll try it out.

That's what I do all the time, quantify the purified product and then use that to work out copy number etc.

Works fine for me.

Just FYI, in an application note by Applied Biosystems: "Amplification Efficiencies of TaqMan Gene Expression Assays" (2004)..


ABI claims that using purified PCR product over a large dynamic range is more reliable for determining efficiency than using the limited amounts of cDNA available after RT.

Ohmyhead-



I would say yes - you want to make sure that the amount of template that you use for your PCR reaction is within your dynamic range, which you can determine by efficiency testing.



If you are adding 10 ng cDNA template to your PCR reactions, I would suggest having your efficiency plot cover something like 0.1, 1, 10, 100, 1000, 10000 ng product or something like that. But honestly, although I have read this information, I have not tried it myself (yet).



One issue I have with this is that it seems natural that purified PCR product would have a higher efficiency regardless of the dynamic range, because it is pure and may not have the inhibitors or other issues that cDNA may have. And even though I can show that efficiency of my PCR conditions is near 100% with pure product, does that mean anything if I can't get PCR of my cDNA to be 100%??



What do others think?

Hi Soluene and OmH,

I wondered about that issue too. I guess that the point of efficiency determination is to compare the efficiency of several genes for RQ. So, things like inhibitors in cDNA should be the same for every gene, right?

I diluted my PCR product 6 fold to do the standard curve, and that was fine - I could have even diluted it 7 or 8 fold and still got usable CT data. The more dilutions you do, the more accurate the curve is, as far as I understand it.

cheers