The importance of selecting the right internal control gene to study the effects of antenatal glucocorticoid administration in human placenta. (August 2016) - Gütling, H.
Abstract: RT-qPCR requires a suitable set of internal control genes (ICGs) for an accurate normalization. The usefulness of 7 previously published ICGs in the human placenta was analyzed according to the effects of betamethasone treatment, sex and fetal age. Raw RT-qPCR data of the ICGs were evaluated using published algorithms. The algorithms revealed that a reliable normalization was achieved using the geometrical mean of PPIA, RPL19, HMBS and SDHA . The use of a different subset ICGs out of the 7 investigated, although not statistically affected by the conditions, biased the results, as demonstrated through changes in expression of glucocorticoid receptor (NR3C1) mRNA as a target gene. Highlights: RT-qPCR requires a suitable set of internal control genes for an accurate normalization. The main effect of antenatal glucocorticoid treatment, gestational age at birth and sex on raw or normalized RT-qPCR was assessed in placenta. Different algorithms (geNorm, BestKeeper, NormFinder) were compared for selecting an appropriate set of internal control genes. Reliable normalization was achieved using the geometrical mean of PPIA, RPL19, HMBS and SDHA accounting for antenatal betamethasone treatment, gestational age and sex.