Elsevier

Methods

Volume 50, Issue 4, April 2010, Pages 298-301
Methods

Analysis of circulating microRNA biomarkers in plasma and serum using quantitative reverse transcription-PCR (qRT-PCR)

https://doi.org/10.1016/j.ymeth.2010.01.032Get rights and content

Abstract

MicroRNAs (miRNAs) are small (∼22 nt) RNAs that play important roles in gene regulatory networks by binding to and repressing the activity of specific target mRNAs. Recent studies have indicated that miRNAs circulate in a stable, cell-free form in the bloodstream and that the abundance of specific miRNAs in plasma or serum can serve as biomarkers of cancer and other diseases. Measurement of circulating miRNAs as biomarkers is associated with some special challenges, including those related to pre-analytic variation and data normalization. We describe here our procedure for qRT-PCR analysis of circulating miRNAs as biomarkers, and discuss relevant issues of sample preparation, experimental design and data analysis.

Introduction

MicroRNAs (miRNAs) are small, non-protein-encoding RNAs that post-transcriptionally regulate gene expression via suppression of specific target mRNAs [1], [2]. Recently, we and others have demonstrated that miRNAs circulate in a highly stable, cell-free form in the blood (i.e., they can be detected in plasma and serum) [3], [4], [5], [6], [7], [8], [9]. Furthermore, tumor cells have been shown to release miRNAs into the circulation [3] and profiles of miRNAs in plasma and serum have been found to be altered in cancer and other disease states [3], [4], [7], [8], suggesting broad opportunities for development of circulating miRNAs as blood-based markers for molecular diagnostics.

Prerequisite to developing circulating miRNA-based diagnostics is the ability to measure miRNAs from plasma and/or serum with sufficient sensitivity and precision to be clinically effective. The small size of the mature miRNA sequence (∼22 nt) and sequence homology between the mature and precursor miRNA forms has required advances in PCR-based detection methods for the quantitative analysis of miRNAs from cultured cells and tissue specimens. These challenges have been met by innovative solutions based on qRT-PCR, as published elsewhere [10], [11], [12].

Adapting miRNA qRT-PCR technology to the analysis of circulating miRNAs, however, requires modified RNA extraction methods to permit use of human plasma or serum as the starting material, dealing with the difficulties in quantifying the minute amounts of RNA typically recovered from plasma or serum, and data normalization to correct for technical variations in the entire procedure. We focus here on discussing these issues, as well as describing a protocol that we have used for circulating miRNA qRT-PCR that is based on commercially available TaqMan technology.

Section snippets

Overview

The clinical effectiveness of circulating miRNAs as biomarkers is likely to be affected by a range of variables including pre-analytic factors having to do with specimen collection and processing, factors influencing RNA extraction efficiency, and the technical issues involved in successful qRT-PCR and data analysis. We will begin with a discussion of issues to be considered at the various steps of this pipeline, followed by a presentation of the detailed protocol that we have used successfully

Concluding remarks

Here we have described one protocol for extraction and quantification of miRNA from human plasma and serum samples that we have found useful in investigating miRNAs as circulating biomarkers. As detailed above, careful matching of case and control samples and pre-analytic steps is critical given the current paucity of knowledge about the influence of pre-analytic variables on miRNA measurements. In addition, careful experimental design in the layout and performance of qRT-PCR is important for

Acknowledgments

M.T. acknowledges support from a Department of Defense (DOD) Prostate Cancer New Investigator Award (PC074012), a Pacific Ovarian Cancer Research Consortium Specialized Program of Research Excellence (SPORE) in Ovarian Cancer Career Development Award (NIH P50 CA83636; N. Urban PI), a Pilot Award from NIH/NCI Cancer Center Support Grant (5 P30 CA015704), a Pacific Northwest Prostate Cancer SPORE Pilot Grant (NIH P50 CA97186; P. Nelson and J. Stanford co-PIs), a DOD Ovarian Cancer Career

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Present address: Molecular and Cellular Biology Graduate Program, University of Washington, Seattle, WA, USA.

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