Protocol for miRNA isolation from biofluids

MicroRNAs (miRNAs) have been identified as promising biomarkers in cancer and other diseases. Packaging of miRNAs into vesicles and complexes with proteins ensures their stability in biological fluids but also complicates their isolation. Conventional protocols used to isolate cell-free RNA are generally successful in overcoming these difficulties; however, they are costly, labor-intensive, or heavily reliant on the use of hazardous chemicals. Here we describe a protocol that is suitable for isolating miRNAs from biofluids, including blood plasma and urine. The protocol is based on precipitation of proteins, denaturation of miRNA-containing complexes with octanoic acid and guanidine isothiocyanate, and subsequent purification of miRNA on spin columns. The efficacy of miRNA extraction by phenol-chloroform extraction, miRCURY RNA isolation kit--biofluids (Exiqon), and the proposed protocol was compared by quantitative reverse-transcription PCR of miR-16 and miR-126. The proposed protocol was slightly more effective for isolating miRNA from plasma and significantly superior to the other two methods for miRNA isolation from urine. Spectrophotometry and SDS-PAGE data suggest that the disparity in performance between miRCURY Biofluids and the proposed protocol can be attributed to differences in precipitation mechanisms, as confirmed by the retention of different proteins in the supernatant.