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

BackgroundBovine 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.MethodsDuring 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.ResultsThree 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.ConclusionsThe 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.

Cultured bovine granulosa cells rapidly lose important features of their identity and functionality but partially recover under long-term culture conditions

“…qPCR was performed with SensiFast SYBR No-ROX (Bioline) and gene-specific primers (see Electronic Supplementary Material, Table S1) in a Light Cycler 96 instrument (Roche, Mannheim, Germany) as described previously (Baddela et al 2014; Yenuganti et al 2016). Normalized qPCR values were then expressed as fold changes relative to the respective transcript abundance found in freshly isolated cells.…”

Human bone marrow stem cells cultured under hypoxic conditions present altered characteristics and enhanced in vivo tissue regeneration

“…The primers specific to each type of differentiation (osteogenic, adipogenic, and chondrogenic) that were used are listed in Table 1. All the relative levels of mRNA expression in this study were normalized to an internal standard gene of B2M, which was known as a stable housekeeping gene regardless of O 2 concentration [18].…”

Overexpression of hypoxia-inducible factor 1α induces migration and invasion through Notch signaling

“…Following the reverse transcription, 1 µl primer (10 µM, summarized in Table I), 12.5 µl SYBRR Premix Ex Taq II, 2 µl cDNA and 8.5 µl dH 2 O mixed together, pre-degeneration for 95˚C, 30 sec, one repeat, and PCR reaction, 95˚C 5 sec followed by 60˚C, 30 sec, 35 repeats, and the dissociation stage, 95˚C, 15 sec followed by 60˚C, 30 sec, and 95˚C, 15 sec, then the data were collected and analyzed. β-actin (25) was used as the internal control. The threshold cycle (Ct) value for triplicate reactions was averaged.…”

“…MG-132 (Sigma) was applied to inhibit the proteasome-dependent degradation if necessary (10 µM, 4 h before the protein harvest) (26). β-actin (25) were used as the loading controls.…”