Comparison of methods for miRNA isolation and quantification from ovine plasma.

ABSTRACT

microRNA (miRNA) are promising candidates for disease biomarkers as they are abundant in circulation, highly stable in biological fluids and may yield diagnostic biomarker signatures. The reported issues with miRNA isolation using traditional RNA reagents necessitates the optimisation of miRNA isolation from challenging samples. In this study we compared six commercial RNA extraction kits to evaluate their ability to isolate miRNA from ovine plasma. We also compared three methods for quantification of small RNA extracted from plasma to determine the most reliable. Using minimal sample inputs of fresh and frozen plasma from five sheep, we compared the six kits (Kit A-F) using quantitative PCR. Operational factors were also assessed for each kit. Kits A and B provided the best detection of the miRNA qPCR reference genes across fresh and frozen samples (p < 0.001) followed by Kit C. The Qubit and microRNA assay provided the least variation (% CV 5.47, SEM ± 0.07), followed by the NanoDrop (% CV 7.01, SEM ± 0.92) and Agilent Bioanalyzer (% CV 59.21, SEM ± 1.31). We identify Kit A to be optimal for isolating miRNA from small volumes of fresh and frozen ovine plasma, and Kit B the top performing kit taking into consideration miRNA detection and operational factors. The Qubit fluorometer using a microRNA assay was the most reliable miRNA quantification method.