PCR efficiencies for analysis of relative real time data - (Nov/27/2008 )
I am in the process of running an experiment with 1 reference gene and 24 target genes on two different templates. I optimised the template concentrations using my reference gene primer set. I then optimised the concentration of the 25 primer sets. However, I did not perform PCR efficiency calculations for all of my 24 target gene primer sets. This was because the amount of starting template that I had was limited (and if I optimised everything with template dilutions, I would not have had enough to do the experiment).
The problem is now I can't analyse my data with either of the programs I was going to use (REST or Q-GENE) as they require PCR efficiency calculations. I am not sure that I have enough template to go back and do the calculations for each of my genes. I am basically looking at absence/presence of the genes in the two tissue types (and in the case where the gene is present in both high/low, but this is less important). I was wondering whether presuming the reaction efficiency is 100% for the analyses is okay in any case (and whether anyone has ever done this or read a paper where it was done). I know that the efficiency calculation effects the accuracy of the calculated expression result, but I am just wondering whether, when there are certain constraints, it is seen as acceptable not to include them? I could probably go back and work out the efficiences for some of the genes that I am looking at, but not all.
Any comments would be appreciated
you can estimate the amplification efficiency from each reaction tube from your raw data. Which might be even more accurate, than assuming that your reaction has always the same efficency.
Have a look at:
Ramakers et al 2003 "Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data" (-->Program called LinReg) or
Tichopad et al 2003 "Standardized determination of real-time PCR efficiency from single reaction set-up"
If you use a Corbett Rotor Cycler you can also get the individual sample efficiency from the Comparative Quantitation function.