Abstract
Selection of reference genes has become an integral step in any real time quantitative PCR (RT-qPCR) based expression studies. The importance of this study stems from the fact that riverine buffaloes are major dairy species of Indian sub-continent and the information generated here will be of great interest to the investigators engaged in functional genomic studies of this important livestock species. In this study, an effort was made to evaluate a panel of 10 candidate reference genes (glyceraldehyde 3-phosphate dehydrogenase (GAPDH), beta- actin (ACTB), ubiquitously expressed transcript (UXT), ribosomal protein S15 (RPS15), ribosomal protein L-4 (RPL4), ribosomal protein S9 (RPS9), ribosomal protein S23 (RPS23), hydroxymethylbilane synthase (HMBS), β2 Microglobulin (β2M) and eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) across 12 tissues (mammary gland, kidney, spleen, liver, heart, intestine, ovary, lung, muscle, brain, subcutaneous fat and testis) of riverine buffaloes. In addition to overall analysis, tissue wise evaluation of expression stability of individual RG was also performed. Three different algorithms provided in geNorm, NormFinder and BestKeeper softwares were used to evaluate the stability of 10 potential reference genes from different functional classes. The M-value given by geNorm ranged from 0.9797 (RPS9 and UXT) to 1.7362 (RPS15). From the most stable to the least stable, genes were ranked as: UXT/RPS9> RPL4> RPS23> EEF1A1> ACTB> HMBS> GAPDH> B2M> RPS15. While NormFinder analysis ranked the genes as: UXT> RPS23> RPL4> RPS9> EEF1A1> HMBS> ACTB> β2M> GAPDH> RPS15. Based on the crossing point SD value and range of fold change expression, BestKeeper analysis ranked the genes as: RPS9> RPS23/UXT> RPL4> GAPDH> EEF1A1> ACTB> HMBS> β2M> RPS15. Overall the study has identified RPS23, RPS9, RPL4 and UXT genes to be the most stable and appropriate RGs that could be utilized for normalization of transcriptional data in various tissues of buffaloes. This manuscript thus provide useful information on panel of reference genes that could be helpful for researchers conducting functional genomic studies in riverine buffaloes.
Highlights
The riverine buffalo (Bubalus bubalis) is major livestock species of India that plays an important role in agricultural economy due to its dairy, meat and draught potential
Not much information is available on set of suitable reference genes that can be used in lactation or physiological studies in riverine buffaloes (Bubalus bubalis) few studies have been performed in specific cell types/tissues [19,20,21]
The quantitative real time polymerase chain reaction (qPCR) performance in terms of slope of five points standard curves was in the range of -3.138 to -3.646 and showed sufficiently good amplification efficiencies ranging from 88.063% for UXT to 109.26% for RPL4
Summary
The riverine buffalo (Bubalus bubalis) is major livestock species of India that plays an important role in agricultural economy due to its dairy, meat and draught potential. 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 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]. Such information will be a useful resource to normalize the qPCR based expression data generated across tissues during various physiological or metabolic studies in riverine buffaloes
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