miRNAs are small non-coding RNA involved in many different biological processes. Their overexpression in cultured cells is an essential tool to study their function.
Here we describe an optimized protocol for transient transfection of miRNAs and siRNAs in combination with plasmid DNA. In this experimental set-up, plasmid DNA is a luciferase reporter that allows quantitative measurements of the biological effect of the overexpressed microRNA on the transcription from a given natural or synthetic promoter. In our paper, this plasmid allowed to read the strength of the TGF-beta pathway activation (pCAGA12-lux).
siRNAs are well known reagents commonly used for gene knock-down; in our experimental set-up siRNA designed against the putative target of an miRNA was used as an experimental positive control and was therefore tested under the same conditions.