Stable reference genes in granulosa cells of bovine dominant follicles during follicular growth, FSH stimulation and maternal aging

Abstract

The aim of the present study was to determine a set of reference genes in granulosa cells of dominant follicles that are suitable for relative gene expression analyses during maternal and follicular aging. Granulosa cells of growing and preovulatory dominant follicles were collected from aged and young cows (maternal aging study) and from FSH-stimulated follicles developing under different durations of FSH treatment (follicular aging study). The mRNA levels of the two commonly used reference genes (GAPDH, ACTB) and four novel genes (UBE2D2, EIF2B2, SF3A1, RNF20) were analysed using cycle threshold values. Results revealed that mRNA levels of GAPDH, ACTB, EIF2B2, RNF20, SF3A1 and UBE2D2 were similar (P > 0.05) between dominant follicle type, age and among follicles obtained after FSH-stimulation, but differed (P = 0.005) due to mRNA processing (i.e. with versus without amplification). The stability of reference genes was analysed using GeNorm, DeltaCT and NormFinder programs and comprehensive ranking order was determined using RefFinder. The mRNA levels of GAPDH and ACTB were less stable than those of UBE2D2 and EIF2B2. The geometric mean of multiple genes (UBE2D2, EIF2B2, GAPDH and SF3A1) is a more appropriate reference control than the use of a single reference gene to compare relative gene expression among dominant and FSH-stimulated follicles during maternal and/or follicular aging studies.