Selection and validation of suitable reference gene for qPCR gene expression analysis in lamb testis cells under Sheep pox virus infection

Abstract

Virus infection alters host gene expression, therefore ideal and stable reference housekeeping genes are required to normalise the expression of other expressed host genes in quantitative real-time PCR (qRT-PCR). The suitable reference gene may vary in response to different viral infections in different hosts or cells. In the present study, we cultured primary lamb testis cells (LTC) and assessed the expression stability of seven widely used housekeeping genes (B2M, HMBS, HPRT1, HSP-90, POLR2A, 18s_RNA, GAPDH) as reference genes in Sheeppox virus (SPPV) infected and control (uninfected-0h) LTC at 0.5h, 4.0h, 8.0h, and 12.0h post-infection) using NormFinder, Bestkeeper, geNorm, and the comparative ΔCT method in RefFinder based on their expression levels. Analysis revealed that HSP90, 18s_RNA, HPRT, POLR2A, and B2M were the most stable genes from the panel in the individual analysis group in 0h, 0.5h, 4.0h, 8.0h, and 12.0h, respectively. Furthermore, B2M was shown to be the most stable reference gene in the combined control with the respective and overall infected groups, except the control group of 4.0hpi of SPPV infection. In this study, we selected the most suitable reference genes in LTC for particular time points of SPPV infection. The identified most suitable housekeeping gene can be used during normalization of expression of other targeted genes at aspecific time point of SPPV infection.