Nested Relative Quatitative Real-Time RT-PCR - (Mar/26/2009 )

Dear Forums,

The title sound complicated... :-)
Basically I am trying to study cytokine gene expression of T cells under unstimulated condition.
The cytokine mRNA level is very low, so low so that is almost undetectable during 1st RT-PCR.
However, if I do a nested PCR by using 1ul of the 1st PCR product..... it works!!
On the othr hand, I am using B-ACT as reference gene which present in high copy number, and I could amplify it
by just doing 1 round of PCR.

So now my question is, since I am runing a relative quatification, thus the exact copy number of
mRNA is not important... Can i just simply devide target gene over the housekeeping gene and I still get a valid result?

Thanks
Hadrian

Hi

I doubt it, because you have to keep in mind that all the calculation you do strongly depend on the reaction efficiency. You can assume it to be 2 or you can estimate it from your data (e.g. 1.85) but I'll guess you will always have a certain failure in this efficiency you calculate with. And this failure is accumulating by cycle number due to the exponential nature of the reaction. That's why you try to set you threshold as early as possible and that's also the reason why your gene of interest and your reference gene should have more or less the same quantity.
So if you know amplify one gene 15 cycles and the other gene 35 cycles I doubt that they are comparable with each other. If you do a two step reaction and don't multiplex I would rather suggest to dilute the cDNA until beta-actin comes at comparable cycle numbers to your cytokine.

Good luck!

hadrian on Mar 27 2009, 01:45 AM said:

Dear WOW,

I am tryng to study IL-4 expression in T cells. Il-4 is know for very low expression and it remains biologically ective in low concentration.
I am done some mistake in the begining of my study that was usign too little cells as starting material. Other researchers were using 5-15 million cells, and i am only using 1/2 million.

I am just wondring, since I am doing relative quatification... if i treat my IL-4 and housekeeping gene as the same (run 2 rounds of PCR 15+40 cycles), should it be compareble?

Thanks

I don't think so.
You see, theoretically, when an amplicon crosses the threshold at 35 cycles it statistically means that you have 1 copy/cell.
After amplifying for 15 cycles, from a single copy you get 8192 (2^13) copies of amplicon. For it to be quantitative, you have to take exactly the same number of copies from the first reaction to the second.

When I did a study on IL-4 we had good results. Did you try other primers?
If you want the reference to the article for the primers we used send a message throw the system.

There are several RNA pre-amplification kits, that transcribe more copies of initial RNA in linear fashion. That is compatible with real-time PCR. Nesting is not.
You can then use lower concentrations of housekeeping primers, if you want to use the same as you're using now or better find another housekeeping gene that has a similar expression as your target genes.