How to Explore the Function and Importance of MicroRNAs: MicroRNAs Expression Profile and Their Target/Pathway Prediction in Bovine Ovarian Cells

Abstract

Micro RNAs (miRNA) are integral components of genetic regulatory networks and act by binding to the transcripts of their corresponding target genes, leading to a decrease in protein production levels either by mRNA degradation or by translational repression. While the role of miRNAs is ubiquitous, they have a particular importance with regard to cell differentiation. The miRNA-target mRNA interaction has a significant impact on many signaling pathways and the cross-talk between them; playing a regulatory role in a variety of different physiological processes within the cells. Ovarian follicle development is a physiological process that is not fully understood with regard to miRNA regulation; there are many questions that remain with respect to the molecular regulation of this important process. Bovine follicular cells are a good experimental model for the investigation of these mechanisms, having direct implications on reproductive health in humans. This chapter describes how differentially expressed miRNAs are identified in the granulosa and theca cells of dominant and subordinate bovine ovarian follicles and the identification of their associated targets and pathways. This chapter systematically describes how the granulosa and theca cells are dissected from the ovarian follicles. Afterward, we present a detailed protocol for miRNA extraction, based on a combined TRI reagent/column clean-up method, and also miRNA expression profiling using both microarray and RT-qPCR. In addition, an outline is provided of the bioinformatic analysis which enables the prediction of miRNAs targets. Pathways associated with the differentially expressed miRNAs are also elucidated using DIANA-miRPath software.