Selection of Suitable Reference Genes for Real-time Quantitative PCR Studies in Lanzhou Fat-tailed Sheep (Ovis aries)

Real-time quantitative PCR (qPCR) is the most accurate method of quantifying gene expression, provided that suitable endogenous controls are used to normalize the data. To date, no reference genes have been validated for development in Lanzhou fat-tailed sheep (Ovis aries). We have determined the expression profiles of 7 housekeeping genes as candidate reference genes (Actb, Ywhaz, Gapdh, Tubb2, Pgk1 and 18S rRNA) in 7 developmental stages (1, 3, 5, 7, 9, 11 and 13 months of age) and 6 tissues (omental fat, liver, tail fat, thigh muscle, subcutaneous fat (backfat above 12th and 13th rib) and perirenal fat) in Lanzhou fat-tailed sheep. The software packages geNorm, NormFinder and BestKeeper were used to evaluate the stability of potential reference genes; each produced comparable results. Initial results showed several of the candidate genes exhibited stable expression throughout development while Actb was identified as the least stable gene. Further analysis with geNorm, NormFinder and BestKeeper identified Gapdh, Tubb2, Sdha and Ywhaz as acceptably stable in gene expression. Comparison of diacylglycerol O-acyltransferase 1 (Dgat1) expression data normalized with geometric averages obtained from combinations of either Gapdh, Sdha, Ywhaz or Tubb2, Sdha, Ywhaz showed no significant differences, indicating that these two combinations are similar. The data provided in this paper may also be useful in guiding researchers performing gene expression in other species of sheep.