A simple method to assess group difference in RT-qPCR reference gene selection using GeNorm: The case of the placental sex

Abstract

Normalization with proper reference genes is a crucial step in obtaining accurate mRNA expression levels in RT-qPCR experiments. GeNorm and NormFinder are two commonly used software packages that help in selecting the best reference genes, based on their expression stability. However, GeNorm does not take into account a group variable, such as sample sex, in its calculation. We demonstrate a simple calculation step to assess the variability of such parameters by multiplying the GeNorm M value with the difference of Cq values between groups. To test this, we used 28 reference gene candidates, to analyze 20 placental samples (10 of each sex), and by using HPRT1 (lower Cq values in male placentas (P = 0.017)), as a target gene. Our calculation demonstrates that the RPL30 - GAPDH reference gene combination is the better option to assess small placental sex differences in mRNA level, versus the selection obtained from GeNorm or NormFinder. The HPRT1 normalized mRNA expression level is different between placental sexes, using RPL30 and GAPDH as reference genes (P = 0.01), but not when using genes suggested by GeNorm or NormFinder. These results indicate that the proposed calculation is appropriate to assess small variations in mRNA expression between 2 groups.