Investigation of gene stability in equine luteal tissue during mid-luteal phase and early pregnancy

Real- time reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a technique that allows for the quantification of mRNA transcripts present within a tissue of interest. To ensure that quantified amounts of mRNA determined by RT qPCR is due to biological differences rather than a product of variation in testing protocol, results need to be normalized. Normalization has historically relied on the use of reference genes, or genes whose transcript expression does not differ in the tissue of interest independent of the experimental condition. In the field of equine reproductive studies, ACTB and GAPDH have been the most widely used reference genes for normalization of RT-qPCR results. However, recent studies have demonstrated that these genes may have drastically varied expression levels in different tissues and in different physiological states. Our study was aimed at examining different putative reference genes (historic reference genes as well as genes identified by RNA-seq to be stable across different sample types) in equine corpus luteum samples at day 11 and day 13 in pregnant and non-pregnant animals. Stability of genetic expression was evaluated via three stability software analyses (GeNorm, NormFinder and BestKeeper). We hypothesized that the most commonly used historic reference genes (ACTB, GAPDH and B2M) would be the most stably expressed genes in equine corpus luteum samples. COX4I1 and SRP14 were both found to be within the top three most stable genes of all samples for all methods. When assessing the least stably expressed genes, the historic reference genes were frequently identified across the three softwares. Exploration of putative reference genes should be considered when investigating dynamic endocrine organs such as those used in reproductive studies. RT-qPCR studies evaluated with historic genes should be interpreted cautiously. Overall design: Healthy normal cycling light breed mares (n=8) were enrolled in the study. A transvaginal luteal biopsy was performed in pregnant and non-pregnant mares at day 11 and day 13 post-ovulation. RNA isolation was performed using TRIzol reagent (ThermoFisher) according to the manufacturer's instructions. RNA libraries were prepared using the NEBNext Ultra II Directional Library prep kit (New England Biolabs). RNA sequencing results were used to identify putative stable reference genes across pregnancy status and timepoint.