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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...
Autores principales: | , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2012
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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. |
format | Online Article Text |
id | pubmed-3496722 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2012 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
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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