Identification of suitable endogenous control genes for quantitative RT-PCR analysis of miRNA in bovine solid tissues.

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) has become the preferred technique for studying low-abundance RNA expression. Proper normalization is a critical but often underappreciated aspect of quantitative gene expression analysis; popular endogenous control genes are usually selected with little knowledge of their real suitability. To date, there are very few reports regarding the general validation of endogenous control genes for microRNA (miRNA) expression analysis in bovine tissue. In the present study, eight candidate reference genes (U6, 18S rRNA, GAPDH, ACTB, miR-191, miR-15a, miR-18a, let-7f) were tested for use as normalizers of bovine miRNA in RT-qPCR assays. Their selection was based on publicly available data concerning normalization, hierarchical clustering and sequencing. Three of the genes (miR-191, U6-1 and let-7f) were found to be highly consistent in their expression across eight different bovine solid tissues. It is commonly accepted that gene expression studies should be normalized using more than one endogenous control gene. Based on our results, we propose using the combined results for miR-191, U6-1 and let-7f as the endogenous control for normalization of miRNA levels in qRT-PCR analysis of diverse bovine tissues. This result could act a guideline for future work on bovine miRNA expression.