Evaluation of housekeeping genes in placental comparative expression studies

Abstract

Preeclampsia and diabetes are complications of pregnancy that contribute to maternal and perinatal mortality worldwide. Results emerging from molecular studies of placentae may elucidate etiologically important genomic alterations. Appropriate application of real time reverse transcription (RT) PCR in comparative gene expression studies requires endogenous housekeeping genes to normalize between sample variations. Ideal housekeeping genes must have stable tissue expression, but few have been specifically studied in the placenta. We sought to identify candidate control genes by analyzing seven functionally distinct housekeeping genes (B2M, GAPDH, HMBS, HPRT, SDHA, TBP, YWHAZ) for their expression stability and level in the placenta. mRNA isolated from 20 placentae was analyzed for gene expression using RT-PCR. Expression stability (M) was assessed using normalization strategies previously used for other tissues. TBP and SDHA were the most stable, with an average expression stability of M = 0.43, followed by YWHAZ (M = 0.44) > HPRT (M = 0.53) > HMBS (M = 0.57) > GAPDH (M = 0.61) > B2M (M = 0.69). The genes tested ranged in abundance, with an approximately 300-fold increase from the lowest (HMBS) to the highest (B2M). By using TBP, SDHA and YWHAZ, with greater expression stability than those housekeeping genes commonly used in placenta studies, gene expression profile comparisons will have more sensitivity and specificity.