ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, UP, India.
ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, UP, India.
Gene. 2022 Jul 15;831:146561. doi: 10.1016/j.gene.2022.146561. Epub 2022 May 11.
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.
病毒感染会改变宿主基因的表达，因此需要理想且稳定的管家基因来对定量实时 PCR（qRT-PCR）中其他表达的宿主基因的表达进行标准化。在不同的宿主或细胞中，针对不同的病毒感染，合适的参考基因可能会有所不同。本研究中，我们培养了原代绵羊睾丸细胞（LTC），并在绵羊痘病毒（SPPV）感染和对照（未感染-0h）的 LTC 中，在感染后 0.5h、4.0h、8.0h 和 12.0h 时，使用 NormFinder、Bestkeeper、geNorm 和 RefFinder 中的比较 ΔCT 方法，评估了七个常用管家基因（B2M、HMBS、HPRT1、HSP-90、POLR2A、18s_RNA 和 GAPDH）作为参考基因的表达稳定性。分析表明，在单独分析组中，HSP90、18s_RNA、HPRT、POLR2A 和 B2M 在 0h、0.5h、4.0h、8.0h 和 12.0h 时是最稳定的基因。此外，B2M 是在分别和总体感染组的联合对照中表现出最稳定的参考基因，除了 SPPV 感染的 4.0hpi 对照组。在本研究中，我们选择了 SPPV 感染特定时间点的 LTC 中最适合的参考基因。所确定的最适管家基因可用于在 SPPV 感染特定时间点对其他靶基因的表达进行归一化。
