Identification of Appropriate Reference Genes for qRT-PCR Analysis of Heat-Stressed Mammary Epithelial Cells in Riverine Buffaloes (Bubalus bubalis)

Abstract

Gene expression studies require appropriate normalization methods for proper evaluation of reference genes. To date, not many studies have been reported on the identification of suitable reference genes in buffaloes. The present study was undertaken to determine the panel of suitable reference genes in heat-stressed buffalo mammary epithelial cells (MECs). Briefly, MEC culture from buffalo mammary gland was exposed to 42 °C for one hour and subsequently allowed to recover at 37 °C for different time intervals (from 30 m to 48 h). Three different algorithms, geNorm, NormFinder, and BestKeeper softwares, were used to evaluate the stability of 16 potential reference genes from different functional classes. Our data identified RPL4, EEF1A1, and RPS23 genes to be the most appropriate reference genes that could be utilized for normalization of qPCR data in heat-stressed buffalo MECs.

Identification of internal control genes in milk‐derived mammary epithelial cells during lactation cycle of Indian zebu cow

“…; Kapila et al . ). The ranking of 10 ICG according to their average expression stability (M value) is shown in Figure and Table .…”

“…Also, RPL4 and EEF1A1 genes were found to be among the most stable genes in buffalo mammary gland transcriptional response to heat stress (Kapila et al . ).…”

Selection of suitable reference genes for normalization of quantitative RT-PCR (RT-qPCR) expression data across twelve tissues of riverine buffaloes (Bubalus bubalis)

“…In recent past, our groups has conducted number of similar Reference genes for qPCR studies across tissues of riverine buffaloes Reference genes for qPCR studies across tissues of riverine buffaloes studies to identify appropriate reference gene panels for application in transcriptional studies in Indian native cattle and riverine buffaloes. In one such study, β2M, RPS9 and RPS15a genes were identified as best RGs for heat stressed mononuclear cells of Indian cattle and buffaloes [19]; β2M, RPS23, RPL4 and EEF1A1 as most reliable RGs in heat stressed mammary explants and mammary epithelial cells of buffaloes [6,7]; RPL4, EEF1A1, ACTB and GAPDH genes were found to be most stable genes in milk derived mammary epithelial cells in Sahiwal cows during different lactation stages [30]. Similarly identification of stable reference genes for transcriptional studies in bulls distinctive in meat quality [5] and in buffalo muscle tissue [31] were also reported.…”

“…The RNA samples were stored at -80˚C till further use. First strand cDNA was synthesized using 100ng RNA as described in our previous studies [7,19] using the program: 25˚C for 5 min, 50˚C for 60 min, and 70˚C for 15 min. cDNA was then diluted 1:4 (v:v) with DNase/RNase free water.…”

“…Use of internal control genes (ICGs) or reference genes (RGs) with constant expression level between samples in response to experimental treatment or physiological state, are now considered as effective method for normalization of transcriptional data to account for the experimental variations. This approach has been widely used in different cell types and tissues in various species [5][6][7]. In past though, trend was to use only single reference gene; mostly GAPDH, ACTB or 18S rRNA to normalize the real time PCR data.…”