Generating Standard for qPCR - PCR vs plasmid DNA (Sep/11/2010 )
I am new in setting up a quantitative PCR experiments and I wish to get some help here. My goal is to quantify the amount of 18S and 16S rRNA in my total RNA samples by using a standard curve method. Does anyone knows what is the different if I were to use purified PCR products (from 18s PCR and 16s PCR) for generating the standard instead of cloning these products into plasmids and generating standard from plasmids?
Help and advice will be greatly appreciated.
Thanks a million.
You can use dilution curve of quantified PCR product, but ends could degrade quickly so it's not good for longer use. I used this (classic PCR with the realtime primers, run on gel, excise the specific product from the gel, purify, measure 260/280, calculate the copy number, dilute) for a one-time experiment, within day or two from the dilution of the standards. But this is most biased method, as you can't account for RT reaction efficiencies and different ratio of target and non-target template. Ideal is to use diluted RT-transcribed RNA of known copy number (with added "dummy" nucleic acid to match the overal concentration, simulating the actual conditions in your sample). But that is hard to get, usualy only few are commercially available.
To create a stable long-term standard it's indeed better to clone the product and then use purified single-cut plasmid diluted in the TE buffer with dummy NA (usualy E.coli rRNA is used, which may be not suitable in your case). This also doesn't account for RT efficiencies, but in my case this is absolute quantification for two genes, that were finaly divided to make a relative ratio, so when the both genes have standards made the same way, this didn't really matter. So it depends what you're going to do with your absolute quantification results.