An Improved Method for Detecting Circulating MicroRNAs with S-Poly(T) Plus Real-time PCR

Overview

Journal Sci Rep

Specialty Science

Date 2015 Oct 14

PMID 26459910

Citations 47

Authors

Yanqin Niu

Limin Zhang

Huiling Qiu

Yike Wu

Zhiwei Wang

Yujia Zai

Lin Liu

Junle Qu

Kang Kang

Deming Gou

Affiliations

Soon will be listed here.

Abstract

We herein describe a simple, sensitive and specific method for analysis of circulating microRNAs (miRNA), termed S-Poly(T) Plus real-time PCR assay. This new method is based on our previously developed S-Poly(T) method, in which a unique S-Poly(T) primer is used during reverse-transcription to increase sensitivity and specificity. Further increased sensitivity and simplicity of S-Poly(T) Plus, in comparison with the S-Poly(T) method, were achieved by a single-step, multiple-stage reaction, where RNAs were polyadenylated and reverse-transcribed at the same time. The sensitivity of circulating miRNA detection was further improved by a modified method of total RNA isolation from serum/plasma, S/P miRsol, in which glycogen was used to increase the RNA yield. We validated our methods by quantifying miRNA expression profiles in the sera of the patients with pulmonary arterial hypertension associated with congenital heart disease. In conclusion, we developed a simple, sensitive, and specific method for detecting circulating miRNAs that allows the measurement of 266 miRNAs from 100 μl of serum or plasma. This method presents a promising tool for basic miRNA research and clinical diagnosis of human diseases based on miRNA biomarkers.

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