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.
Highlights
The riverine buffaloes exhibit signs of great distress when exposed to direct solar radiations
The total RNA extracted from individual mammary epithelial cells (MECs) samples exhibited high purity as determined by mean (±SEM) A260/280 ratio of 2.06 ± 0.014
The analysis identified the same set of genes (RPL4, eukaryotic translation elongation factor 1 alpha 1 (EEF1A1), GTP-binding protein (GTP), ubiquitously expressed transcript (UXT), ribosomal protein S23 (RPS23)) being most stable as revealed by geNorm only with slight change in their ranking order (RPL4 > EEF1A1 > GTP > UXT > RPS23) (Table 4, Figure 4)
Summary
The riverine buffaloes (bubalus bubalis) exhibit signs of great distress when exposed to direct solar radiations. QPCR is a common tool to determine the expression level of any target gene; for accurate quantification of expression level, there is a need to identify the appropriate reference genes under the particular experimental setup Such approaches are helping a great deal to normalize the real time data for reliable interpretation of expression studies in different species [4,5,6,7,8]. A number of studies have been conducted to identify the stably expressed candidate genes in different tissues of various livestock species such as pig, sheep, and bovines [5, 16,17,18,19] These reports suggested that expression levels of commonly used reference genes vary considerably between different tissues and experimental conditions. A total of 16 known reference genes, namely, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), betaactin (ACTB), ubiquitously expressed transcript (UXT), ribosomal protein S15A (RPS15A), beta 2-microglobulin (B2M), alpha 2-microglobulin (A2M), ribosomal protein L4 (RPL4), ribosomal proteinS18 (RS18), ribosomal protein L22 (RPL22), ribosomal protein S9 (RPS9), ribosomal protein S23 (RPS23), hydroxymethylbilane synthase (HMBS), hypoxanthine-guanine phosphoribosyltransferase (HPRT1), GTP-binding protein (GTP), eukaryotic translation elongation factor 1 alpha 1 (EEF1A1), and ubiquitin C (UBC) belonging to different functional categories, were evaluated (Table 1)
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