19 - A single molecule method to quantify miRNA gene expression

Summary

Introduction

Deemed the “breakthrough of the year” by Science magazine in 2002, research into the biology of small RNA regulation has grown exponentially in recent years; however, the field is relatively nascent in terms of identifying and characterizing the universe of miRNAs and their expression in various biological states. According to the miRNA registry (release 6.0, www.sanger.ac.uk/Software/Rfam/mirna/index.shtml); of the 319 predicted human miRNAs the expressions of 234 have been experimentally verified by Northern blot, cloning, or microarray. Further, the total number of miRNAs within a genome is unknown. Thus, sensitive, specific, quantitative, and rapid methods for measuring the expression levels of miRNAs would significantly advance the field.

The short 21 nucleotide nature of these molecules makes them difficult to study via conventional techniques. They are not easily amplified which makes miRNA microarrays and quantitative PCR technically challenging. Despite these challenges, several groups have undertaken miRNA microarray studies to quantify miRNA gene expression. Their approaches are similar in requiring up-front enrichment for small RNAs, reverse transcription, PCR amplification, labeling, and clean-up steps. While the arrays are superior at large scale screening they lack the ability to finely discriminate expression levels and are at best semi-quantitative. Theoretically the most sensitive technique to quantify miRNAs is reverse transcription RT-PCR (real time RT-PCR). However, this method is difficult in both assay (probe) design and execution. Tissue samples must be devoid of enzyme inhibitors to enable efficient reverse transcription and amplification steps (Tichopad et al., 2004).