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A Novel Real-Time PCR Assay of microRNAs Using S-Poly(T), a Specific Oligo(dT) Reverse Transcription Primer with Excellent Sensitivity and Specificity

BACKGROUND: MicroRNAs (miRNAs) are small, non-coding RNAs capable of postranscriptionally regulating gene expression. Accurate expression profiling is crucial for understanding the biological roles of miRNAs, and exploring them as biomarkers of diseases. METHODOLOGY/PRINCIPAL FINDINGS: A novel, high...

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Autores principales: Kang, Kang, Zhang, Xiaoying, Liu, Hongtao, Wang, Zhiwei, Zhong, Jiasheng, Huang, Zhenting, Peng, Xiao, Zeng, Yan, Wang, Yuna, Yang, Yi, Luo, Jun, Gou, Deming
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3496722/
https://www.ncbi.nlm.nih.gov/pubmed/23152780
http://dx.doi.org/10.1371/journal.pone.0048536
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author Kang, Kang
Zhang, Xiaoying
Liu, Hongtao
Wang, Zhiwei
Zhong, Jiasheng
Huang, Zhenting
Peng, Xiao
Zeng, Yan
Wang, Yuna
Yang, Yi
Luo, Jun
Gou, Deming
author_facet Kang, Kang
Zhang, Xiaoying
Liu, Hongtao
Wang, Zhiwei
Zhong, Jiasheng
Huang, Zhenting
Peng, Xiao
Zeng, Yan
Wang, Yuna
Yang, Yi
Luo, Jun
Gou, Deming
author_sort Kang, Kang
collection PubMed
description BACKGROUND: MicroRNAs (miRNAs) are small, non-coding RNAs capable of postranscriptionally regulating gene expression. Accurate expression profiling is crucial for understanding the biological roles of miRNAs, and exploring them as biomarkers of diseases. METHODOLOGY/PRINCIPAL FINDINGS: A novel, highly sensitive, and reliable miRNA quantification approach,termed S-Poly(T) miRNA assay, is designed. In this assay, miRNAs are subjected to polyadenylation and reverse transcription with a S-Poly(T) primer that contains a universal reverse primer, a universal Taqman probe, an oligo(dT)(11) sequence and six miRNA-specific bases. Individual miRNAs are then amplified by a specific forward primer and a universal reverse primer, and the PCR products are detected by a universal Taqman probe. The S-Poly(T) assay showed a minimum of 4-fold increase in sensitivity as compared with the stem-loop or poly(A)-based methods. A remarkable specificity in discriminating among miRNAs with high sequence similarity was also obtained with this approach. Using this method, we profiled miRNAs in human pulmonary arterial smooth muscle cells (HPASMC) and identified 9 differentially expressed miRNAs associated with hypoxia treatment. Due to its outstanding sensitivity, the number of circulating miRNAs from normal human serum was significantly expanded from 368 to 518. CONCLUSIONS/SIGNIFICANCE: With excellent sensitivity, specificity, and high-throughput, the S-Poly(T) method provides a powerful tool for miRNAs quantification and identification of tissue- or disease-specific miRNA biomarkers.
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spelling pubmed-34967222012-11-14 A Novel Real-Time PCR Assay of microRNAs Using S-Poly(T), a Specific Oligo(dT) Reverse Transcription Primer with Excellent Sensitivity and Specificity Kang, Kang Zhang, Xiaoying Liu, Hongtao Wang, Zhiwei Zhong, Jiasheng Huang, Zhenting Peng, Xiao Zeng, Yan Wang, Yuna Yang, Yi Luo, Jun Gou, Deming PLoS One Research Article BACKGROUND: MicroRNAs (miRNAs) are small, non-coding RNAs capable of postranscriptionally regulating gene expression. Accurate expression profiling is crucial for understanding the biological roles of miRNAs, and exploring them as biomarkers of diseases. METHODOLOGY/PRINCIPAL FINDINGS: A novel, highly sensitive, and reliable miRNA quantification approach,termed S-Poly(T) miRNA assay, is designed. In this assay, miRNAs are subjected to polyadenylation and reverse transcription with a S-Poly(T) primer that contains a universal reverse primer, a universal Taqman probe, an oligo(dT)(11) sequence and six miRNA-specific bases. Individual miRNAs are then amplified by a specific forward primer and a universal reverse primer, and the PCR products are detected by a universal Taqman probe. The S-Poly(T) assay showed a minimum of 4-fold increase in sensitivity as compared with the stem-loop or poly(A)-based methods. A remarkable specificity in discriminating among miRNAs with high sequence similarity was also obtained with this approach. Using this method, we profiled miRNAs in human pulmonary arterial smooth muscle cells (HPASMC) and identified 9 differentially expressed miRNAs associated with hypoxia treatment. Due to its outstanding sensitivity, the number of circulating miRNAs from normal human serum was significantly expanded from 368 to 518. CONCLUSIONS/SIGNIFICANCE: With excellent sensitivity, specificity, and high-throughput, the S-Poly(T) method provides a powerful tool for miRNAs quantification and identification of tissue- or disease-specific miRNA biomarkers. Public Library of Science 2012-11-13 /pmc/articles/PMC3496722/ /pubmed/23152780 http://dx.doi.org/10.1371/journal.pone.0048536 Text en © 2012 Kang et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Kang, Kang
Zhang, Xiaoying
Liu, Hongtao
Wang, Zhiwei
Zhong, Jiasheng
Huang, Zhenting
Peng, Xiao
Zeng, Yan
Wang, Yuna
Yang, Yi
Luo, Jun
Gou, Deming
A Novel Real-Time PCR Assay of microRNAs Using S-Poly(T), a Specific Oligo(dT) Reverse Transcription Primer with Excellent Sensitivity and Specificity
title A Novel Real-Time PCR Assay of microRNAs Using S-Poly(T), a Specific Oligo(dT) Reverse Transcription Primer with Excellent Sensitivity and Specificity
title_full A Novel Real-Time PCR Assay of microRNAs Using S-Poly(T), a Specific Oligo(dT) Reverse Transcription Primer with Excellent Sensitivity and Specificity
title_fullStr A Novel Real-Time PCR Assay of microRNAs Using S-Poly(T), a Specific Oligo(dT) Reverse Transcription Primer with Excellent Sensitivity and Specificity
title_full_unstemmed A Novel Real-Time PCR Assay of microRNAs Using S-Poly(T), a Specific Oligo(dT) Reverse Transcription Primer with Excellent Sensitivity and Specificity
title_short A Novel Real-Time PCR Assay of microRNAs Using S-Poly(T), a Specific Oligo(dT) Reverse Transcription Primer with Excellent Sensitivity and Specificity
title_sort novel real-time pcr assay of micrornas using s-poly(t), a specific oligo(dt) reverse transcription primer with excellent sensitivity and specificity
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3496722/
https://www.ncbi.nlm.nih.gov/pubmed/23152780
http://dx.doi.org/10.1371/journal.pone.0048536
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