Establishment of platform for quantifying circulating miRNAs in clinical serum samples

Objective To establish reliable platform for quantifying circulating miRNA in clinical serum or plasma samples. Methods Serum（plasma） samples were collected routinely, and total RNA was extracted using mirVana PARIS kit and recovered total RNA preparations were treated with DNase I. Mamm small nuclear U6 and target miRNAs in total RNA preparation were reverse transcribed by specific stem-loop primers and real-time fluorescence quantitative polymerase chain reaction（rt-fqPCR） was adopted to quantify U6 and target miRNAs. Results The total RNA concentrations extracted from 10 fresh plasma samples were 3.5 to 35.4ng/μl. U6, miR-16 and miR-224 were amplified and quantified specifically in RNA preparations isolated from 400μl routinely collected clinical serum samples, with corresponding Ct values of 30, 25 and 32, respectively. Furthermore, total RNA were extracted from 6 serum samples maintained at room temperature for different time and the corresponding RNA levels were 10.24ng/μl and 4.46ng/μl, and quantitative PCR results indicated the abundance of miR-16 and miR-224 were stable. Conclusion Extracting total RNA from clinical serum or plasma samples are indeed feasible and target miRNAs in clinical serum（plasma） samples can be readily detected by real-time fqPCR with high sensitivity and specificity.