Haemolysis during Sample Preparation Alters microRNA Content of Plasma

Michaela B Kirschner,J James Edelman,Nico Van Zandwijk,Nicola J Armstrong,Glen Reid,Michael P Vallely,Steven C Kao,Sebastien Pfeffer

doi:10.1371/journal.pone.0024145

Abstract

The presence of cell-free microRNAs (miRNAs) has been detected in a range of body fluids. The miRNA content of plasma/serum in particular has been proposed as a potential source of novel biomarkers for a number of diseases. Nevertheless, the quantification of miRNAs from plasma or serum is made difficult due to inefficient isolation and lack of consensus regarding the optimal reference miRNA. The effect of haemolysis on the quantification and normalisation of miRNAs in plasma has not been investigated in great detail. We found that levels of miR-16, a commonly used reference gene, showed little variation when measured in plasma samples from healthy volunteers or patients with malignant mesothelioma or coronary artery disease. Including samples with evidence of haemolysis led to variation in miR-16 levels and consequently decreased its ability to serve as a reference. The levels of miR-16 and miR-451, both present in significant levels in red blood cells, were proportional to the degree of haemolysis. Measurements of the level of these miRNAs in whole blood, plasma, red blood cells and peripheral blood mononuclear cells revealed that the miRNA content of red blood cells represents the major source of variation in miR-16 and miR-451 levels measured in plasma. Adding lysed red blood cells to non-haemolysed plasma allowed a cut-off level of free haemoglobin to be determined, below which miR-16 and miR-451 levels displayed little variation between individuals. In conclusion, increases in plasma miR-16 and miR-451 are caused by haemolysis. In the absence of haemolysis the levels of both miR-16 and miR-451 are sufficiently constant to serve as normalisers.

Highlights

MicroRNAs, a class of 18–25 nt long non-coding RNAs, are post-transcriptional modulators of gene expression [1,2,3]
A number of studies have shown that miRNAs are readily detectable in body fluids, and the presence of specific miRNA patterns in plasma of diseased patients has raised the possibility of their use as biomarkers [7,8,9]
Since the first reports revealing the presence of miRNAs in plasma and serum, numerous studies have identified distinct miRNA expression patterns associated with disease and have proposed them as candidate biomarkers [9]

Summary

Introduction

MicroRNAs (miRNAs), a class of 18–25 nt long non-coding RNAs, are post-transcriptional modulators of gene expression [1,2,3]. They are involved in the regulation of normal physiological processes and there is rapidly increasing evidence that they play a prominent role in cancer [2,4,5] and non-malignant conditions such as heart disease [6]. A number of studies have shown that miRNAs are readily detectable in body fluids, and the presence of specific miRNA patterns in plasma of diseased (cancer) patients has raised the possibility of their use as biomarkers [7,8,9]. When comparing the methods applied in different studies, a consensus on the best methods for the measurement and accurate quantification of disease-related miRNA patterns in body fluids has yet to be reached

Methods

Results

Conclusion