Improved Detection of Circulating miRNAs in Serum and Plasma Following Rapid Heat/Freeze Cycling

摘要

Background: The measurement of circulating miRNAs has proven to be a powerful biomarkertool for several disease processes. Current protocols for the detection of miRNAs usually involvean RNA extraction step, requiring a substantial volume of patient serum or plasma to obtain sufficientinput material.Objective: Here, we describe a novel methodology that allows detection of a large number of miRNAsfrom a small volume of serum or plasma without the need for RNA extraction.Methods: Three μl of serum or plasma was subjected to three cycles of high and low temperatures(heat/freeze cycles) followed by miRNA arrays.Results: Our results indicate that miRNA detection following this process is highly reproducible whencomparing multiple samples from the same subject. Moreover, this protocol increases the reproducibilityof miRNA detection in samples that were previously subjected to multiple freeze-thaw cycles.Importantly, the detection of miRNAs from serum vs. plasma following heat/freeze cycling are highlycomparable, indicating that this heat/freeze process effectively eliminates differences in detection betweenserum and plasma samples that have been reported using other sample preparation methodologies.Conclusion: We propose that this method is a potent alternative to current RNA extraction protocols,substantially reducing the amount of sample necessary for miRNA detection while simultaneously improvingmiRNA detection and reproducibility.