Detection and quantification of extracellular microRNAs in murine biofluids

Thomas C Roberts,Matthew J A Wood,Corinne A Betts,Anna M L Coenen-Stass

doi:10.1186/1480-9222-16-5

Thomas C Roberts, Matthew J A Wood + Show 2 more

Open Access

https://doi.org/10.1186/1480-9222-16-5

Copy DOI

Abstract

BackgroundMicroRNAs (miRNAs) are short RNA molecules which regulate gene expression in eukaryotic cells, and are abundant and stable in biofluids such as blood serum and plasma. As such, there has been heightened interest in the utility of extracellular miRNAs as minimally invasive biomarkers for diagnosis and monitoring of a wide range of human pathologies. However, quantification of extracellular miRNAs is subject to a number of specific challenges, including the relatively low RNA content of biofluids, the possibility of contamination with serum proteins (including RNases and PCR inhibitors), hemolysis, platelet contamination/activation, a lack of well-established reference miRNAs and the biochemical properties of miRNAs themselves. Protocols for the detection and quantification of miRNAs in biofluids are therefore of high interest.ResultsThe following protocol was validated by quantifying miRNA abundance in C57 (wild-type) and dystrophin-deficient (mdx) mice. Important differences in miRNA abundance were observed depending on whether blood was taken from the jugular or tail vein. Furthermore, efficiency of miRNA recovery was reduced when sample volumes greater than 50 μl were used.ConclusionsHere we describe robust and novel procedures to harvest murine serum/plasma, extract biofluid RNA, amplify specific miRNAs by RT-qPCR and analyze the resulting data, enabling the determination of relative and absolute miRNA abundance in extracellular biofluids with high accuracy, specificity and sensitivity.

Highlights

MicroRNAs are short RNA molecules which regulate gene expression in eukaryotic cells, and are abundant and stable in biofluids such as blood serum and plasma
MicroRNAs are short RNA molecules involved in gene regulation in higher organisms [1] which have recently been detected in extracellular biofluids including serum/plasma [2,3], urine [4], cerebral spinal fluid [5], saliva [6] and seminal fluid [7]
We have recently shown that there is a tendency for serum from the dystrophin-deficient mdx mouse to have a higher miRNA and total RNA content than serum from wild-type non-dystrophic mice [42]

Summary

Introduction

MicroRNAs (miRNAs) are short RNA molecules which regulate gene expression in eukaryotic cells, and are abundant and stable in biofluids such as blood serum and plasma. MicroRNAs (miRNAs) are short RNA molecules involved in gene regulation in higher organisms [1] which have recently been detected in extracellular biofluids including serum/plasma [2,3], urine [4], cerebral spinal fluid [5], saliva [6] and seminal fluid [7]. Since their discovery in 1993 [8] miRNAs have been the subject of intense study on account of their involvement in a plethora of physiological and pathophysiological processes. Whether or not extracellular miRNAs can be taken up by tissues, and thereby transfer gene regulatory information between cells, is currently a matter for debate and an exciting area of ongoing research [10,11,23,24]

Methods

Results

Conclusion