Abstract
MicroRNAs (miRNAs) can exhibit aberrant expression under different physiological and pathological conditions. Therefore, differentially expressed circulating miRNAs have been a focus of biomarker discovery research. However, the use of circulating miRNAs comes with challenges which may hinder the reliability for their clinical application. These include varied sample collection protocols, storage times/conditions, sample processing and analysis methods. This study focused on examining the effect of whole blood holding time on the stability of plasma miRNA expression profiles. Whole blood samples were collected from healthy pregnant women and were held at 4°C for 30 min, 2 h, 6 h or 24 h prior to processing for plasma isolation. Plasma RNA was extracted and the expression of 179 miRNAs were analyzed. Unsupervised principal component analysis demonstrated that whole blood holding time was a major source of variation in miRNA expression profiles with 53 of 179 miRNAs showing significant changes in expression. Levels of specific miRNAs previously reported to be associated with pregnancy-associated complications such as hsa-miR-150-5p, hsa-miR-191-5p, and hsa-miR-29a-3p, as well as commonly used endogenous miRNA controls, hsa-miR-16-5p, hsa-miR-25-3p, and hsa-miR-223-3p were significantly altered with increase in blood holding time. Current protocols for plasma-based miRNA profiling for diagnostics describe major differences in whole blood holding periods ranging from immediately after collection to 26 h after. Our results demonstrate holding time can have dramatic effects on analytical reliability and reproducibility. This highlights the importance of standardization of blood holding time prior to processing for plasma in order to minimize introduction of non-biological variance in miRNA profiles.
Highlights
MicroRNAs are small single-stranded RNA molecules of approximately 19–22 nucleotides in size that regulate gene expression at post-transcriptional level (Cook et al, 2019)
This study investigated the effect of whole blood holding time prior to plasma processing on level/risk of hemolysis using three of these methods including visual
Some previous studies report high stability of plasma miRNAs for up to 72 h after collection (Mitchell et al, 2008; McDonald et al, 2011), whereas others state that blood holding time up to 4 h significantly affected the levels of specific miRNAs (Wu et al, 2016)
Summary
MicroRNAs (miRNAs) are small single-stranded RNA molecules of approximately 19–22 nucleotides in size that regulate gene expression at post-transcriptional level (Cook et al, 2019). Despite the presence of RNases in blood, miRNAs remain stable and protected from degradation by RNA-binding proteins (Arroyo et al, 2011), specific lipoproteins (Vickers et al, 2011), or via encapsulation within exosomes (Valadi et al, 2007). This high level of stability and the relative ease of accessing miRNAs through blood sampling has led to increased interest in their utility as non-invasive biomarkers for diagnosis, prognosis and prediction of a wide range of pathological conditions. This study aimed to focus on the effects of whole blood holding time, that is storage time until processing for plasma isolation, on miRNA expression profiles
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