which standards... - (Jun/13/2007 )

Hi there real timers!

I've never worked with real-time pcr before, so please go easy with the newbie.

I want to use real-time pcr for different things, but both for quantification and I'm a little bit confused on which and how I should build my standard curve.

So, first thing first. I'm using a cell line model, in which I would like to quantify the level of expression (mRNA) of different genes before and after treatment. What would be more suitable to use as a standard?

First I was thinking in extracting total DNA and run a "normal" pcr with my qPCR primers (they span exon-exon junction, so they don't amplify gDNA). Run the product of that pcr in a agarose gel, cut the band, quantify and dilute into standards. The problem is that probably the efficiency of my standards is going to be allot higher then my samples, since it doesn't have all the other DNA do disturb the reaction. How can I circumvent this issue, or is there any other way?

I'm also constructing a stable reporter cell line and I would like to do a quantification in the same manner, before and after. But in these case, since what I want to quantify is the level of mRNA of the plasmid inserted in the genome, I was thinking in using the plasmid itself as a standard. But then I would run into the same problem....right!?

Thanks in advance.

First, I you want to compare two different cell lines with each other you don't actually need standards like in absolute quantification, because what you want is relative amount and differences between cell lines (before treatment, after treatment).

What you need is pcr for the gene of interest and another pcr for a gene called "housekeeping" e.g. that is expressed on the same level without influence of the treatment (you need to determine a suitable housekeeper or use more of them, to ensures the level stays the same).
Housekeepers are used to normalize your gene of interest (for example that the initial amount of cDNA is different in different samples, different RT efficiency etc.).

Then, if you want to use a Pfaffl method, you need to determine pcr efficiency for both the housekeeper pcr and your gene. This is done by a serial dilution of untreated sample for example, you only need to state relative amounts, you only need the standard curve slope.

For RT real-time it is indeed unsuitable to use DNA standards, because the efficiency is different. If needed for absolute quantification (like detection of RNA viruses) a RNA standards of known concentration must be made, but that is tricky.

Good tutorial for real-time here.

Hope it's understandable, I just looked into this last month myself