RNA expression calculations - (Feb/09/2007 )
I am using Taqman PCR to determine the expression of several mRNAs. Is there any reason real time data should not be calculated as % housekeeping gene transcripts (e.g. % B-actin) rather than as fold expression relative to a calibrator (comparative CT method)?
That is, calculate (2-dCT)*100 rather than 2-ddCT
Also, can %B-actin values be used to compare the transcript levels between different mRNAs? Assuming 100% efficiency of all primer/probes.
well if you trust your curves a lot, than you can work directly on the dCt values, but you should not do any kind of statistic on the %values. You can, however, compare dCT values of your gene between different treatments, using t-tests or ANOVA (depending on the number of groups) or, if you have a small n, non-parametric statistics (Mann-Whitney-U or Kruskal-Wallis).
But, the ddCT method surely is more accurate!
You can give it a try - a good indicator if you're on the safe side may be a very small variance in the deltaCT value of all samples treated the same.