Abstract
Cell-free microRNAs in plasma and serum have become a promising source of biomarkers for various diseases. Despite rapid progress in this field, there remains a lack of consensus regarding optimal quantification methods, reference genes, and quality control of samples. Recent studies have shown that hemolysis occurring during blood collection has substantial impact on the microRNA content in plasma/serum. To date, the impact of hemolysis has only been investigated for a limited number of microRNAs, mainly the red blood cell (RBC)-enriched miRs-16 and -451. In contrast, the effect of hemolysis on other microRNAs – in particular those proposed as biomarkers – has not been addressed. In this study we profiled the microRNA content of hemolyzed and non-hemolyzed plasma as well as RBCs to obtain a profile of microRNAs in the circulation affected or unaffected by hemolysis. Profiling by TaqMan Array Microfluidic Cards was used to compare three pairs of hemolyzed and non-hemolyzed plasma (with varying degrees of hemolysis) and one RBC sample. A total of 136 microRNAs were detectable in at least two of the samples, and of those 15 were at least twofold elevated in all three hemolyzed samples. This number increased to 88 microRNAs for the sample with the highest level of hemolysis, with all of these also detected in the RBC profile. Thus these microRNAs represent a large proportion of detectable microRNAs and those most likely to be affected by hemolysis. Several of the hemolysis-susceptible microRNAs (e.g., miRs-21, -106a, -92a, -17, -16) have also been previously proposed as plasma/serum biomarkers of disease, highlighting the importance of rigorous quality control of plasma/serum samples used for measurement of circulating microRNAs. As low-level hemolysis is a frequent occurrence during plasma/serum collection it is critical that this is taken into account in the measurement of any candidate circulating microRNA.
Highlights
Since the discovery that microRNAs are present within cells, but can be detected extracellularly in a variety of body fluids, a large number of studies have investigated the potential use of these cell-free microRNAs as diagnostic and/or prognostic biomarkers (Reid et al, 2011; Creemers et al, 2012; Mo et al, 2012)
The number of microRNAs detected with a Cq < 35 in each sample varied between 91 and 194, as did the number of microRNAs affected by hemolysis (Figure 1A)
Cell-free microRNAs are detectable in a variety of body fluids and have in recent years attracted a lot of attention due to their potential use as biomarkers for disease
Summary
Since the discovery that microRNAs are present within cells, but can be detected extracellularly in a variety of body fluids, a large number of studies have investigated the potential use of these cell-free microRNAs as diagnostic and/or prognostic biomarkers (Reid et al, 2011; Creemers et al, 2012; Mo et al, 2012) Unlike their longer mRNA counterparts which are prone to degradation in body fluids as a result of the high content of RNase in blood, microRNAs are surprisingly stable. Our own data (Kirschner et al, 2011) and those of Pritchard et al (2012) revealed that levels of miR92a, a microRNA proposed as a potential biomarker for ischemic heart disease (Fichtlscherer et al, 2010) and various cancer types (Tanaka et al, 2009; Huang et al, 2010; Ohyashiki et al, 2011)
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