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Pre-amplification as a method for improvement of quantitative RT-PCR analysis of circulating miRNAs

INTRODUCTION: The assessment of circulating miRNAs is challenging and still limited due to their low concentrations, small size and lack of reference values in human biological samples. Pre-amplification of complementary DNAs may facilitate reliable miRNA quantification. The aim of our study was to...

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Autores principales: Sekovanić, Ankica, Dorotić, Adrijana, Jurasović, Jasna, Pašalić, Daria, Kovačić, Jelena, Stasenko, Sandra, Mioč, Tatjana, Piasek, Martina, Orct, Tatjana
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Croatian Society of Medical Biochemistry and Laboratory Medicine 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7745165/
https://www.ncbi.nlm.nih.gov/pubmed/33380895
http://dx.doi.org/10.11613/BM.2021.010901
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author Sekovanić, Ankica
Dorotić, Adrijana
Jurasović, Jasna
Pašalić, Daria
Kovačić, Jelena
Stasenko, Sandra
Mioč, Tatjana
Piasek, Martina
Orct, Tatjana
author_facet Sekovanić, Ankica
Dorotić, Adrijana
Jurasović, Jasna
Pašalić, Daria
Kovačić, Jelena
Stasenko, Sandra
Mioč, Tatjana
Piasek, Martina
Orct, Tatjana
author_sort Sekovanić, Ankica
collection PubMed
description INTRODUCTION: The assessment of circulating miRNAs is challenging and still limited due to their low concentrations, small size and lack of reference values in human biological samples. Pre-amplification of complementary DNAs may facilitate reliable miRNA quantification. The aim of our study was to evaluate the efficacy of pre-amplification as a step to increase the sensitivity of qPCR analysis for five candidate circulating miRNAs presumably related to toxic metals and cigarette smoke exposure: miR-1537, miR-190b, miR-16, miR-21, and miR-146a. MATERIALS AND METHODS: Candidate miRNAs expression was analysed in plasma samples of 19 mother-newborn pairs. For isolation, transcription, pre-amplification and qPCR quantification kits and protocols by Qiagen (Hilden, Germany) were used. Paired t-test or Wilcoxon rank test were used to compare miRNAs expression levels with and without a pre-amplification step prior to qPCR, separately in maternal and cord plasma. Intraclass correlation (ICC) was calculated as an agreement measure between procedures for each miRNA. RESULTS: Pre-amplification facilitated the detection of all assayed miRNAs with an overall cycle threshold (C(T)) improvement of 6.6 ± 0.89 (P < 0.05). Excellent ICCs (> 0.90) were found between data for preamplified and not preamplified miR-16, miR-21 and miR-146a. However, these correlations for low expressed miR-190b were moderate (0.79 in maternal; 0.61 in cord plasma) and poor for miR-1537 (0.49 in maternal; no correlation in cord plasma). CONCLUSION: Pre-amplification is a useful, necessary step in the analysis of miR-1537 and miR-190b as a reliable procedure facilitating extracellular miRNA expression detection in human plasma by real-time PCR quantification.
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spelling pubmed-77451652020-12-29 Pre-amplification as a method for improvement of quantitative RT-PCR analysis of circulating miRNAs Sekovanić, Ankica Dorotić, Adrijana Jurasović, Jasna Pašalić, Daria Kovačić, Jelena Stasenko, Sandra Mioč, Tatjana Piasek, Martina Orct, Tatjana Biochem Med (Zagreb) Short Communications INTRODUCTION: The assessment of circulating miRNAs is challenging and still limited due to their low concentrations, small size and lack of reference values in human biological samples. Pre-amplification of complementary DNAs may facilitate reliable miRNA quantification. The aim of our study was to evaluate the efficacy of pre-amplification as a step to increase the sensitivity of qPCR analysis for five candidate circulating miRNAs presumably related to toxic metals and cigarette smoke exposure: miR-1537, miR-190b, miR-16, miR-21, and miR-146a. MATERIALS AND METHODS: Candidate miRNAs expression was analysed in plasma samples of 19 mother-newborn pairs. For isolation, transcription, pre-amplification and qPCR quantification kits and protocols by Qiagen (Hilden, Germany) were used. Paired t-test or Wilcoxon rank test were used to compare miRNAs expression levels with and without a pre-amplification step prior to qPCR, separately in maternal and cord plasma. Intraclass correlation (ICC) was calculated as an agreement measure between procedures for each miRNA. RESULTS: Pre-amplification facilitated the detection of all assayed miRNAs with an overall cycle threshold (C(T)) improvement of 6.6 ± 0.89 (P < 0.05). Excellent ICCs (> 0.90) were found between data for preamplified and not preamplified miR-16, miR-21 and miR-146a. However, these correlations for low expressed miR-190b were moderate (0.79 in maternal; 0.61 in cord plasma) and poor for miR-1537 (0.49 in maternal; no correlation in cord plasma). CONCLUSION: Pre-amplification is a useful, necessary step in the analysis of miR-1537 and miR-190b as a reliable procedure facilitating extracellular miRNA expression detection in human plasma by real-time PCR quantification. Croatian Society of Medical Biochemistry and Laboratory Medicine 2020-12-15 2021-02-15 /pmc/articles/PMC7745165/ /pubmed/33380895 http://dx.doi.org/10.11613/BM.2021.010901 Text en Croatian Society of Medical Biochemistry and Laboratory Medicine. This is an Open Access article distributed under the terms of the Creative Commons Attribution (http://creativecommons.org/licenses/by/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Short Communications
Sekovanić, Ankica
Dorotić, Adrijana
Jurasović, Jasna
Pašalić, Daria
Kovačić, Jelena
Stasenko, Sandra
Mioč, Tatjana
Piasek, Martina
Orct, Tatjana
Pre-amplification as a method for improvement of quantitative RT-PCR analysis of circulating miRNAs
title Pre-amplification as a method for improvement of quantitative RT-PCR analysis of circulating miRNAs
title_full Pre-amplification as a method for improvement of quantitative RT-PCR analysis of circulating miRNAs
title_fullStr Pre-amplification as a method for improvement of quantitative RT-PCR analysis of circulating miRNAs
title_full_unstemmed Pre-amplification as a method for improvement of quantitative RT-PCR analysis of circulating miRNAs
title_short Pre-amplification as a method for improvement of quantitative RT-PCR analysis of circulating miRNAs
title_sort pre-amplification as a method for improvement of quantitative rt-pcr analysis of circulating mirnas
topic Short Communications
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7745165/
https://www.ncbi.nlm.nih.gov/pubmed/33380895
http://dx.doi.org/10.11613/BM.2021.010901
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