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Universal Stem-Loop Primer Method for Screening and Quantification of MicroRNA

RT-qPCR is the accepted technique for the quantification of microRNA (miR) expression: however, stem-loop RT-PCR, the most frequently used method for quantification of miRs, is time- and reagent-consuming as well as inconvenient for scanning. We established a new method called ‘universal stem-loop p...

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Autores principales: Yang, Li-hong, Wang, Si-lu, Tang, Li-li, Liu, Biao, Ye, Wen-le, Wang, Ling-ling, Wang, Zhang-yang, Zhou, Meng-tao, Chen, Bi-cheng
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4280144/
https://www.ncbi.nlm.nih.gov/pubmed/25548906
http://dx.doi.org/10.1371/journal.pone.0115293
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author Yang, Li-hong
Wang, Si-lu
Tang, Li-li
Liu, Biao
Ye, Wen-le
Wang, Ling-ling
Wang, Zhang-yang
Zhou, Meng-tao
Chen, Bi-cheng
author_facet Yang, Li-hong
Wang, Si-lu
Tang, Li-li
Liu, Biao
Ye, Wen-le
Wang, Ling-ling
Wang, Zhang-yang
Zhou, Meng-tao
Chen, Bi-cheng
author_sort Yang, Li-hong
collection PubMed
description RT-qPCR is the accepted technique for the quantification of microRNA (miR) expression: however, stem-loop RT-PCR, the most frequently used method for quantification of miRs, is time- and reagent-consuming as well as inconvenient for scanning. We established a new method called ‘universal stem-loop primer’ (USLP) with 8 random nucleotides instead of a specific sequence at the 3′ end of the traditional stem-loop primer (TSLP), for screening miR profile and to semi-quantify expression of miRs. Peripheral blood samples were cultured with phytohaemagglutinin (PHA), and then 87 candidate miRs were scanned in cultured T cells. By USLP, our study revealed that the expression of miR-150-5p (miR-150) decreased nearly 10-fold, and miR-155-5p (miR-155) increased more than 7-fold after treated with PHA. The results of the dissociation curve and gel electrophoresis showed that the PCR production of the USLP and TSLP were specificity. The USLP method has high precision because of its low ICV (ICV<2.5%). The sensitivity of the USLP is up to 10(3) copies/µl miR. As compared with the TSLP, USLP saved 75% the cost of primers and 60% of the test time. The USLP method is a simple, rapid, precise, sensitive, and cost-effective approach that is suitable for screening miR profiles.
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spelling pubmed-42801442015-01-07 Universal Stem-Loop Primer Method for Screening and Quantification of MicroRNA Yang, Li-hong Wang, Si-lu Tang, Li-li Liu, Biao Ye, Wen-le Wang, Ling-ling Wang, Zhang-yang Zhou, Meng-tao Chen, Bi-cheng PLoS One Research Article RT-qPCR is the accepted technique for the quantification of microRNA (miR) expression: however, stem-loop RT-PCR, the most frequently used method for quantification of miRs, is time- and reagent-consuming as well as inconvenient for scanning. We established a new method called ‘universal stem-loop primer’ (USLP) with 8 random nucleotides instead of a specific sequence at the 3′ end of the traditional stem-loop primer (TSLP), for screening miR profile and to semi-quantify expression of miRs. Peripheral blood samples were cultured with phytohaemagglutinin (PHA), and then 87 candidate miRs were scanned in cultured T cells. By USLP, our study revealed that the expression of miR-150-5p (miR-150) decreased nearly 10-fold, and miR-155-5p (miR-155) increased more than 7-fold after treated with PHA. The results of the dissociation curve and gel electrophoresis showed that the PCR production of the USLP and TSLP were specificity. The USLP method has high precision because of its low ICV (ICV<2.5%). The sensitivity of the USLP is up to 10(3) copies/µl miR. As compared with the TSLP, USLP saved 75% the cost of primers and 60% of the test time. The USLP method is a simple, rapid, precise, sensitive, and cost-effective approach that is suitable for screening miR profiles. Public Library of Science 2014-12-30 /pmc/articles/PMC4280144/ /pubmed/25548906 http://dx.doi.org/10.1371/journal.pone.0115293 Text en © 2014 Yang 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
Yang, Li-hong
Wang, Si-lu
Tang, Li-li
Liu, Biao
Ye, Wen-le
Wang, Ling-ling
Wang, Zhang-yang
Zhou, Meng-tao
Chen, Bi-cheng
Universal Stem-Loop Primer Method for Screening and Quantification of MicroRNA
title Universal Stem-Loop Primer Method for Screening and Quantification of MicroRNA
title_full Universal Stem-Loop Primer Method for Screening and Quantification of MicroRNA
title_fullStr Universal Stem-Loop Primer Method for Screening and Quantification of MicroRNA
title_full_unstemmed Universal Stem-Loop Primer Method for Screening and Quantification of MicroRNA
title_short Universal Stem-Loop Primer Method for Screening and Quantification of MicroRNA
title_sort universal stem-loop primer method for screening and quantification of microrna
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4280144/
https://www.ncbi.nlm.nih.gov/pubmed/25548906
http://dx.doi.org/10.1371/journal.pone.0115293
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