Identification and validation of reference genes for gene expression studies in water buffalo

In gene expression analysis, a key step to obtain informative data from reverse transcription quantitative PCR (RT qPCR) assay is normalization, that is usually achieved by ratio to correct the abundance of the gene of interest against that of an endogenous reference gene. The finding of such reference genes, ideally expressed in a stable way in multiple tissue samples and in different experimental conditions, is a non-trivial problem. In this work, a set of genes potentially useful as reference for gene expression studies in water buffalo has been identified and evaluated. In the first step, a publicly available Bos taurus expressed sequence tags database has been downloaded from the TIGR Gene Index and mined by some simple frequency algorithms to find out which tentative consensuses are present in a remarkable number of different cDNA libraries and, consequently, are more suitable to be included in a starter set of candidate reference genes. To validate the potential of such candidates for their use as normalizers in buffalo gene expression analysis, an RT qPCR analysis has been carried out, in which the expression stability of these genes has been evaluated on a panel of buffalo tissues and organs. Our results indicate that ribosomal proteins L4 and L5 and Gek protein encoding genes can be useful as normalizers to compare gene expression levels across tissues and organs in buffalo.