A reporter gene imaging system for monitoring microRNA biogenesis

Abstract

MicroRNAs (miRNAs), non-coding RNA molecules, have emerged as a part of key gene regulation, participating in a variety of biological processes such as cell development. Current research methods, including northern blot and real-time PCR analysis, have been used to quantify miRNA expression. Major disadvantages of these methods include invasive techniques, such as a tissue biopsy, and the absence of repetitive studies. In this protocol we describe a simple non-invasive imaging method for monitoring miRNAs during neurogenesis. This novel method includes the design of an miRNA reporter gene vector, cell transfection, in vitro luciferase assay and in vivo bioluminescence imaging of miRNAs. Our reporter imaging system allows for repetitive, non-invasive detection of miRNAs, illustrating the miRNA124a (miR124a)-dependent decrease of Gaussia reporter activity during neuronal differentiation. Using this method, construction of a reporter-imaging vector, in vitro and in vivo signal detection steps can be carried out in similar to 10 d.