Determination of the optimal number of control genes for normalization. Pairwise variation (Vn(n+1)) analysis between the normalization factors (NF
) and (NFn+1) to determine the number of control genes required for accurate normalization. Normalization factors were computed taking 3–11 most stable genes for all the three cell lines. Pairwise variation of 0.15 was taken as a cut off value . For A431 and BxPC3 three ECGs were sufficient for normalization in contrast to six ECGs for A549.