ORIGINAL ARTICLE Identification of internal control genes in milk-derived mammary epithelial cells during lactation cycle of Indian zebu cow Pradeep JATAV, 1 Monika SODHI, 1 Ankita SHARMA, 1 Sandeep MANN, 1 Amit KISHORE, 1 Umesh K SHANDILYA, 1 Ashok K MOHANTY, 2 Ranjit S KATARIA, 1 Poonam YADAV, 1 Preeti VERMA, 1 Surinder KUMAR, 2 Dhruba MALAKAR 2 and Manishi MUKESH 1 1 National Bureau of Animal Genetic Resources and 2 National Dairy Research Institute, Haryana, India 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. Key words: gene expression, internal control gene, lactation, mammary epithelial cells, qPCR. INTRODUCTION Gene expression analysis is an important tool in many fields of biological research. Understanding expressed gene patterns is critical to provide insights into complex regulatory networks and identification of genes rel- evant to new biological processes (Vandesompele et al. 2002). There is a growing interest in understanding the physiology of lactation in dairy animals, particularly to explore which genes control the composition of milk and how these genes are regulated. Two recently devel- oped methods, microarrays and quantitative polymer- ase chain reaction (qPCR) are the most commonly used techniques to analyze gene expression patterns. Among the array of techniques available, qPCR is the most sensitive and popular technique for messenger RNA (mRNA) quantification. Compared to Northern blot analysis and ribonuclease (RNase) protection assay, qPCR can be used to quantify mRNA levels even from small quantity of samples. In fact, this technique is sensitive enough to enable quantification of RNA from a single cell. It has also become the preferred method to validate the results obtained from microarray analyses. To date, expression kinetics of important genes regulat- ing milk production is poorly understood in Indian zebu cattle (Bos indicus). The major limitation in understand- ing the lactation biology of Indian cows is getting the mammary biopsies from the live experimental animals. Due to various ethical and religious issues, the invasive biopsy procedures are not being performed on a routine basis in Indian cattle. To overcome this difficulty, milk- derived mammary epithelial cells (MECs) could be employed as an alternative source for gene expression studies. As MECs are responsible for converting most precursors into milk constituents and transporting them to the mammary lumen, these cells isolated from Correspondence: Manishi Mukesh, Principal Scientist and ICAR National Fellow, National Bureau of Animal Genetic Resources, Karnal-132001, Haryana, India. (Email: mmukesh_26@hotmail.com; mmukesh26@gmail.com) Received 10 July 2014; accepted for publication 11 Decem- ber 2014. doi: 10.1111/asj.12384 © 2016 Japanese Society of Animal Science Animal Science Journal (2016) 87, 344–353