One-phase phenol-free method for microRNA isolation from blood plasma

Abstract

MicroRNA extraction is an essential procedure when discovering MicroRNA-based biomarkers and approaches. Here we describe a new method for microRNA isolation from human blood plasma, based on isopropanol precipitation from the one-phase lysate. We demonstrate that the use of more than four volumes of lysis buffer based on 5 M guanidine isothiocyanate prevents the formation of large, viscous, and hardly soluble precipitate. Applying widely used linear polyacrylamide (LPAA) as the only precipitating agent proved ineffective. At the same time, adding poly(A)RNA or tRNA with LPAA significantly increased the amount of microRNA obtained. Replacing β-mercaptoethanol with less volatile dithiothreitol in lysis buffer did not lead to a decrease in the yield. We compared the method proposed with miRNeasy Mini Kit (Qiagen) for isolation of microRNA from human blood plasma. MicroRNA yield was evaluated by the difference in median Ct values obtained for exogenous cel-238 and endogenous microRNA-21 cDNA amplification. For both tested microRNA, the precipitation from one-phase lysate provided better recovery with lower Ct values (Δ median Ct 4.94 for cel-238, р = 1,0E-04 and Δ median Ct 2.18 for microRNA-21, р = 9,0E-04). Thus, the method we described showed high yield and operating convenience because it can be applied without special equipment. Protocol name: One-phase phenol-free method for microRNA isolation from blood plasma, Keywords: Short RNA, Isolation, Biofluids, Protocol name: One-phase phenol-free method for microRNA isolation from blood plasma