Team:Heidelberg/Project/miRNA Kit

Abstract

Introduction

MicroRNAs (miRNAs) are endogenous 22-nt long non-coding RNAs that regulate gene expression post-transcriptionally in a diversity of organisms (Bartel, 2004). Although the understanding of miRNA biological functions is progressing remarkably, the exact mechanisms by which miRNAs regulate gene expression are still not unambiguously defined. However, it is commonly believed that miRNAs trigger target mRNA regulation by binding to 3’UTRs (Chekuleva & Filipowicz, 2009). The discovery of the first miRNA (lin-4) revealed sequence complementarity to multiple conserved sites in the 3’UTR of the lin-14 mRNA (Lee et al., 1993; Wightman et al., 1993). Exact principles of gene expression knockdown mediated by miRNA are still in debate (Eulalio et al., 2008).
Binding site properties have an essential impact on miRNA-mRNA interaction. Rational design of binding sites could thereby help to achieve a narrow range of knockdown. The applicability is still limited by redundant target sites and various miRNA expression patterns within the cells which are hardly to measure. The experimental identification and/or validation of miRNA targets by means of proteomic or transcriptomic studies, reporter gene assays or over-expression/knockdown analysis is necessary to understand the biological relevance of the miRNA-mRNA interaction. Although progressively improving, even the reliability of in silico target prediction tools is still far away from being precise.

Results

Discussion

Methods

Since the miTuner was constructed initially for Dual-Luciferase assay, this was the method of choice for tuning quantification:

• Dual Luciferase Assay

References

Bruce A. Sherf, Shauna L. Navarro,Rita R. Hannah and Keith V. Dua l-LuciferaseTM Reporter Assay: An Advanced Co-Reporter Technology Integrating Firefly and Renilla Luciferase Assays. WoodPromega Notes Magazine Number 57, 1996, p.02

Consumables and Reagents

LumaPlate, PerkinElmer, catalogue number 6005630 Promega Dual-Luciferase® Reporter Assay System, catalogue number E1910

Instruments

Promega GLOMAX 96 Microplate Luminometer

References