An improved method for detecting circulating microRNAs with S-Poly(T) Plus real-time PCR.

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.

journal_name

Sci Rep

journal_title

Scientific reports

authors

Niu Y,Zhang L,Qiu H,Wu Y,Wang Z,Zai Y,Liu L,Qu J,Kang K,Gou D

doi

10.1038/srep15100

subject

Has Abstract

pub_date

2015-10-13 00:00:00

pages

15100

issn

2045-2322

pii

srep15100

journal_volume

5

pub_type

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