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Two-tailed RT-qPCR: a novel method for highly accurate miRNA quantification
MicroRNAs are a class of small non-coding RNAs that serve as important regulators of gene expression at the posttranscriptional level. They are stable in body fluids and pose great potential to serve as biomarkers. Here, we present a highly specific, sensitive and cost-effective system to quantify m...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5587787/ https://www.ncbi.nlm.nih.gov/pubmed/28911110 http://dx.doi.org/10.1093/nar/gkx588 |
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author | Androvic, Peter Valihrach, Lukas Elling, Julie Sjoback, Robert Kubista, Mikael |
author_facet | Androvic, Peter Valihrach, Lukas Elling, Julie Sjoback, Robert Kubista, Mikael |
author_sort | Androvic, Peter |
collection | PubMed |
description | MicroRNAs are a class of small non-coding RNAs that serve as important regulators of gene expression at the posttranscriptional level. They are stable in body fluids and pose great potential to serve as biomarkers. Here, we present a highly specific, sensitive and cost-effective system to quantify miRNA expression based on two-step RT-qPCR with SYBR-green detection chemistry called Two-tailed RT-qPCR. It takes advantage of novel, target-specific primers for reverse transcription composed of two hemiprobes complementary to two different parts of the targeted miRNA, connected by a hairpin structure. The introduction of a second probe ensures high sensitivity and enables discrimination of highly homologous miRNAs irrespectively of the position of the mismatched nucleotide. Two-tailed RT-qPCR has a dynamic range of seven logs and a sensitivity sufficient to detect down to ten target miRNA molecules. It is capable to capture the full isomiR repertoire, leading to accurate representation of the complete miRNA content in a sample. The reverse transcription step can be multiplexed and the miRNA profiles measured with Two-tailed RT-qPCR show excellent correlation with the industry standard TaqMan miRNA assays (r(2) = 0.985). Moreover, Two-tailed RT-qPCR allows for rapid testing with a total analysis time of less than 2.5 hours. |
format | Online Article Text |
id | pubmed-5587787 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-55877872017-09-11 Two-tailed RT-qPCR: a novel method for highly accurate miRNA quantification Androvic, Peter Valihrach, Lukas Elling, Julie Sjoback, Robert Kubista, Mikael Nucleic Acids Res Methods Online MicroRNAs are a class of small non-coding RNAs that serve as important regulators of gene expression at the posttranscriptional level. They are stable in body fluids and pose great potential to serve as biomarkers. Here, we present a highly specific, sensitive and cost-effective system to quantify miRNA expression based on two-step RT-qPCR with SYBR-green detection chemistry called Two-tailed RT-qPCR. It takes advantage of novel, target-specific primers for reverse transcription composed of two hemiprobes complementary to two different parts of the targeted miRNA, connected by a hairpin structure. The introduction of a second probe ensures high sensitivity and enables discrimination of highly homologous miRNAs irrespectively of the position of the mismatched nucleotide. Two-tailed RT-qPCR has a dynamic range of seven logs and a sensitivity sufficient to detect down to ten target miRNA molecules. It is capable to capture the full isomiR repertoire, leading to accurate representation of the complete miRNA content in a sample. The reverse transcription step can be multiplexed and the miRNA profiles measured with Two-tailed RT-qPCR show excellent correlation with the industry standard TaqMan miRNA assays (r(2) = 0.985). Moreover, Two-tailed RT-qPCR allows for rapid testing with a total analysis time of less than 2.5 hours. Oxford University Press 2017-09-06 2017-07-13 /pmc/articles/PMC5587787/ /pubmed/28911110 http://dx.doi.org/10.1093/nar/gkx588 Text en © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com |
spellingShingle | Methods Online Androvic, Peter Valihrach, Lukas Elling, Julie Sjoback, Robert Kubista, Mikael Two-tailed RT-qPCR: a novel method for highly accurate miRNA quantification |
title | Two-tailed RT-qPCR: a novel method for highly accurate miRNA quantification |
title_full | Two-tailed RT-qPCR: a novel method for highly accurate miRNA quantification |
title_fullStr | Two-tailed RT-qPCR: a novel method for highly accurate miRNA quantification |
title_full_unstemmed | Two-tailed RT-qPCR: a novel method for highly accurate miRNA quantification |
title_short | Two-tailed RT-qPCR: a novel method for highly accurate miRNA quantification |
title_sort | two-tailed rt-qpcr: a novel method for highly accurate mirna quantification |
topic | Methods Online |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5587787/ https://www.ncbi.nlm.nih.gov/pubmed/28911110 http://dx.doi.org/10.1093/nar/gkx588 |
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