Assessment of isomiR Discrimination Using Commercial qPCR Methods.

Eric Londin,Tess Cherlin,Isidore Rigoutsos,Rogan Magee,Aristeidis G. Telonis

doi:10.3390/ncrna3020018

Abstract

We sought to determine whether commercial quantitative polymerase chain reaction (qPCR) methods are capable of distinguishing isomiRs: variants of mature microRNAs (miRNAs) with sequence endpoint differences. We used two commercially available miRNA qPCR methods to quantify miR-21-5p in both synthetic and real cell contexts. We find that although these miRNA qPCR methods possess high sensitivity for specific sequences, they also pick up background signals from closely related isomiRs, which influences the reliable quantification of individual isomiRs. We conclude that these methods do not possess the requisite specificity for reliable isomiR quantification.

Highlights

MicroRNAs are a well characterized family of short non-coding RNAs [1,2]
With the use of the vast resource of The Cancer Genome Atlas (TCGA), we were able to take a closer look at the expression of the miR-21-5p locus across 10,274 samples representing 32 distinct tissue types
This is further highlighted in the lower panel of the figure, which shows that the miR-21-5p 0|0 isomiR is expressed over a large range of values in each of the 32 tissues we surveyed

Summary

Introduction

MicroRNAs (miRNAs) are a well characterized family of short non-coding RNAs [1,2] These regulatory molecules are cleaved from premature transcripts by Dicer family proteins to form ~22 nucleotide (nt) molecules. The use of short RNA sequencing has revealed that a miRNA precursor constitutively produces a cloud of mature miRNAs from a given precursor arm, instead of only one. These isomiRs differ from the archetypal sequence (the one annotated in public reference databases such as miRBase [2]) on their 5 - and/or -3 end. We showed that isomiRs can distinguish amongst tissue type and disease subtypes and that isoforms of the same miRNA can target largely non-overlapping groups of mRNAs [10,16]

Methods

Results

Conclusion