Selection of reliable reference genes for gene expression studies involving peripheral blood mononuclear cells in small ruminants

Abstract

Quantitative PCR (qPCR) is a highly sensitive, cost effective, and routinely used molecular assay for analyzing the gene expression patterns of specific target genes across tissues, pathological conditions, treatment regimes, and physiological states. However, normalization of expression profiles of target genes using stable reference genes (RGs) is a critical step to ensure the accuracy of relative quantification using qPCR. In this study, we evaluated the stability of fourteen potential existing reference genes (ACTB, BACH1, B2M, GAPDH, HMBS, PGK1, PPIA, PPIB, RPLP0, RPL19, RPS9, RPS15, RPS28, and UXT) in the peripheral blood mononuclear cells (PBMCs) of healthy sheep and goats to determine the most stable RGs. These candidate genes belong to different functional classes and were chosen based on published literature on commonly used RGs in different livestock species. Four different analytical approaches (geNorm, NormFinder, BestKeeper, and ΔCt analysis) as well as RefFinder, an online tool which integrates the geometric means of these four prominent stability algorithms were utilized to determine a comprehensive ranking of the investigated genes. Our data indicates that PPIB, BACH1, ACTB, and PPIA are the most suitable RGs, while RPLP0, GAPDH and RPS15 are the most variable and unsuitable genes for normalization of qPCR data in the PBMCs of sheep and goats. The results of this study provide useful resource for researchers engaged in unravelling the transcriptional landscape of PBMCs of small ruminants for various scientific investigations.