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Sci Rep. 2015 Oct 13;5:15100. doi: 10.1038/srep15100.

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

Author information

1
Shenzhen Key Laboratory of Microbial Genetic Engineering, Shenzhen Key Laboratory of Marine Bioresource and Eco-environmental Science, College of Life Sciences, Shenzhen University, Shenzhen, Guangdong, 518060, China.
2
Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, College of Optoelectronic Engineering Shenzhen University, Shenzhen, Guangdong, 518060, China.
3
Department of Cardiovascular Surgery, Shenzhen Sun Yat-Sen Cardiovascular Hospital, Shenzhen, Guangdong, 518060, China.
4
Department of Physiological Sciences, Oklahoma State University, Stillwater, OK 74048, USA.
5
Department of Physiology, Shenzhen University Health Science Center, Shenzhen, Guangdong, 518000, China.

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.

PMID:
26459910
PMCID:
PMC4602224
DOI:
10.1038/srep15100
[Indexed for MEDLINE]
Free PMC Article

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