comparative Ct method and statistical significance test - (Jan/06/2009 )
I am comparing the expression of a gene using the comparative Ct method following the “Guide to Perform Relative Quantitation of Gene Expression using Real-Time Quantitative PCR from Applied Biosystem.
I did the experiment two times (biological replicate 1 and 2). For each bio replicate two separate assays were done and duplicate of the cDNA was included during each run (i.e. four technical replicates). Thus for each gene a total of eight Ct values were obtained. My questions is on the presentation of the data
1)Mean and SD values: when is best to combine the two biological replicates? Before or after ΔCt calculation? Or they should be treated separately and combine the 2-ΔΔCt values at last? In the later case how shall I compute the SD?
2)Statistical significance test: I found that different opinion on using a raw Ct (Yuan JS et al., 2006) Vs transformed 2-ΔΔCt (Schmittgen TD and Livak KJ, 2001 and 2008) values to calculate p-values. Which one you think I have to follow?
We take the average and Standard deviations of the biological replicates after 2-ΔΔCt calculation.
Thanks newarray and then how you calculate the SD? Directly from 2-ΔΔCt?