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. 1. Introduction The riverine buffaloes (bubalus bubalis) exhibit signs of great distress when exposed to direct solar radiations. This is generally attributed to their specific morphological, anatomical, and behavioural characteristics [1]. The effect of heat stress on mammary epithelial cell (MEC), the major cell type in lactating mammary gland, could be one of the prime factors responsible for lower milk production in animals. Understanding of expression profile of these cells from different livestock species during different physiological stages would provide molecular basis of heat stress specific transcriptomic response of mammary gland. Recently, few efforts [2, 3] have been made to unravel the transcriptional response of mammary gland to heat stress condition; still, the molecular mechanism of such responses are thought to be too complex to understand. 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 [4–8]. In earlier studies, researchers have relied mostly on GAPDH, ACTB, and RS18 as suitable reference genes [9–15]. However, Vandesompele et al. and Bustin et al. had shown that use of single reference gene can lower the reliability of expression data and strongly advocated use of multiple reference genes for each experimental setup [10, 12]. A number of studies have been conducted to identify the stably expressed candidate genes in different tissues of
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