Suitable housekeeping genes for normalization of transcript abundance analysis by real-time RT-PCR in cultured bovine granulosa cells during hypoxia and differential cell plating density

Abstract

Background: Bovine granulosa cell culture models are important to understand molecular mechanisms of ovarian function. Folliculogenesis and luteinization are associated with increasing density of cells and local hypoxic conditions. The current study identified two reliable housekeeping genes useful for gene normalization in granulosa cells under different in vitro conditions.

Methods: During the current experiments cells were subjected to different biological and physical stimuli, follicle stimulating hormone, different initial cell plating density and hypoxia. Transcript abundance of seven housekeeping genes was quantified by real-time RT-PCR with co-amplification of the respective external standard.

Results: Three of the genes, GAPDH, HMBS, and HPRT1 were found to be regulated by initial cell plating density, five of them, GAPDH, HMBS, HPRT1, RPLP0 and RPS18 under hypoxic conditions, but none of them after FSH stimulation. In detail, GAPDH was up regulated, but HPRT1 and HMBS were down regulated at high density and under hypoxia. Expression of RPLP0 and RPS18 was inconsistent, but was significantly down-regulated in particular at high cell density combined with hypoxia. In contrast, TBP and B2M genes were neither regulated under different plating density conditions nor by hypoxia as they showed similar expression levels under all conditions analyzed.

Conclusions: The present data indicate that TBP and B2M are appropriate housekeeping genes for normalization of transcript abundance measured by real-time RT-PCR in granulosa cells subjected to different plating densities, oxygen concentrations and FSH stimulation.

Figure 1

Effects of FSH and different cell plating density on transcript abundance in cultured bovine granulosa cells. The expression of GAPDH (a), B2M (b), TBP (c), HMBS (d), RPS18 (e), RPLP0 (f) and HPRT1 (g) was analyzed under FSH treatment at low and high initial cell plating density. Copy numbers were calculated and normalized to initial amounts of RNA used for cDNA preparation. Values are means ± SEMs of three independent cultures. Bars with different letters are significantly different from each other.

Figure 2

Effects of hypoxic and normoxic conditions and of different cell plating density on transcript abundance in cultured bovine granulosa cells. The expression of GAPDH (a), B2M (b), TBP (c), HMBS (d), RPS18 (e), RPLP0 (f) and HPRT1 (g) was analyzed under hypoxic (5% O₂) and normoxic conditions (20% O₂) at low and high cell plating density. Copy numbers were calculated and normalized to initial amounts of RNA used for cDNA preparation. Values are means ± SEMs of three independent cultures. Bars with different letters are significantly different from each other.