临床血清循环miRNAs定量技术的建立

Establishment of platform for quantifying circulating miRNAs in clinical serum samples

目的建立可靠的临床血清(浆)循环miRNAs定量技术。方法常规收集血清(浆)标本,mirVana PARIS试剂盒法抽提血清(浆)总RNA,采用DNaseI消化总RNA提取液,以miRNAs特异性茎-环引物引导反转录,通过TaqMan实时荧光定量PCR对U6及靶miRNAs进行检测。结果10份新鲜血浆总RNA浓度介于3.5～35.4ng/μl之间。对常规收集的400μl临床血清标本中U6、miR-16、miR-224均能实现特异扩增及定量,相应的平均Ct值约为30、25及32。6份不同留置时间血清标本总RNA浓度分别10.24和4.46ng/μl,定量PCR结果显示其中相应miR-16和miR-224的丰度却相对稳定。结论血清(浆)总RNA抽提及循环miRNAs定量切实可行。

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.