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

Abstract

The present study aims to evaluate the suitability of 10 candidate genes, namely GAPDH, ACTB, RPS15A, RPL4, RPS9, RPS23, HMBS, HPRT1, EEF1A1 and UBI as internal control genes (ICG) to normalize the transcriptional data of mammary epithelial cells (MEC) in Indian cows. A total of 52 MEC samples were isolated from milk of Sahiwal cows (major indigenous dairy breed of India) across different stages of lactation: Early (5-15 days), Peak (30-60 days), Mid (100-140 days) and Late (> 240 days). Three different statistical algorithms: geNorm, Normfinder and BestKeeper were used to assess the suitability of these genes. In geNorm analysis, all the genes exhibited expression stability (M) values below 0.5 with EEF1A1 and RPL4 showing the maximum expression stability. Similar to geNorm, Normfinder also identified EEF1A1 and RPL4 as two of the most stable genes. In Bestkeeper algorithm as well, all the 10 genes showed consistent expression levels. The analysis showed that four genes, that is, EEF1A1, RPL4, GAPDH and ACTB exhibited higher coefficient of correlation to the Bestkeeper index, lower coefficient of variance and standard deviation, indicating their superiority to be used as ICG. The present analysis has provided evidence that RPL4, EEF1A1, GAPDH and ACTB could probably act as most suitable genes for normalizing the transcriptional data of milk-derived mammary epithelial cells of Indian cows.

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.…”

Heat stress modulates differential response in skin fibroblast cells of native cattle (Bos indicus) and riverine buffaloes (Bubalus bubalis)

“…A dissociation protocol with an incremental temperature of 95 • C for 60 s, 55 • C for 30 s and 95 • C for 30 s was used to investigate the specificity of qPCR reaction and the presence of primer dimers. Further expression data were normalized with three internal control genes: U6 snRNA, S18, and β5s-RNA and compared using first derivative method [14].…”

Identification of suitable internal control genes for transcriptional studies in Eleusine coracana under different abiotic stress conditions

“…Quantitative real-time PCR (qRT-PCR) is one of the most powerful and reliable techniques to quantify gene expression. This technique is widely used due to its sensitivity, accuracy and reproducibility in gene expression analysis (Artico et al 2010;Jatav et al 2016). Quantification assays are performed to detect differential expression of a gene(s) involved in plant growth, development, signal transduction and metabolism.…”