Identification of suitable housekeeping genes for expression analysis in mammary epithelial cells of buffalo (Bubalus bubalis) during lactation cycle

The aim of the research reported herein was to evaluate the stability of 7 frequently used housekeeping genes including ACTB (β-actin), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ubiquitously expressed transcript protein (UXT), ribosomal protein S9 (RPS9), ribosomal protein S15 (RPS15), ribosomal protein S23 (RPS23) and hypoxanthine-guanine phosphoribosyl transferase (HPRT) in mammary epithelial cells (MECs) isolated from buffalo milk at different phases of lactation (15 d, 30 d, 45 d, 60 d, 120 d and 240 d relative to parturition) of three buffaloes. The expression of the genes varied considerably in different MECs samples analyzed. GAPDH showed the highest expression, whereas the expression of UXT was the lowest in MECs of buffalo during all stages of lactation. Microsoft excel based visual application i.e. geNORM and Normfinder were used to rank candidate reference genes based on expression stability. RPS9 and RPS23 were found to be the most stable genes during lactation in buffalo. Geometric mean of these genes can be used for normalization of real time PCR data in mammary epithelial cells during lactation.