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Impact of different stabilization methods on RT-qPCR results using human lung tissue samples

Aiming to increase the reproducibility of biomedical research results, biobanks obtain human tissues of the highest quality and carry out different storage methods adapted to the needs of analytical technique to be performed by the biomedical researchers. However, there is much controversy and littl...

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Autores principales: Esteva-Socias, Margalida, Gómez-Romano, Fernando, Carrillo-Ávila, José Antonio, Sánchez-Navarro, Alicia Loreto, Villena, Cristina
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7046779/
https://www.ncbi.nlm.nih.gov/pubmed/32108147
http://dx.doi.org/10.1038/s41598-020-60618-x
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author Esteva-Socias, Margalida
Gómez-Romano, Fernando
Carrillo-Ávila, José Antonio
Sánchez-Navarro, Alicia Loreto
Villena, Cristina
author_facet Esteva-Socias, Margalida
Gómez-Romano, Fernando
Carrillo-Ávila, José Antonio
Sánchez-Navarro, Alicia Loreto
Villena, Cristina
author_sort Esteva-Socias, Margalida
collection PubMed
description Aiming to increase the reproducibility of biomedical research results, biobanks obtain human tissues of the highest quality and carry out different storage methods adapted to the needs of analytical technique to be performed by the biomedical researchers. However, there is much controversy and little data concerning the real impact of different stabilization methods on tissue quality, integrity and functionality of derived biomolecules. The influence of four stabilization methods [RNAlater (RNL), snap freezing (SF), snap freezing using Optimal Cutting Tissue compound (SF-OCT) and formalin-fixed paraffin-embedded (FFPE)] on RNA quality and integrity was evaluated in paired samples of lung tissue. RNA integrity was evaluated through PCR-endpoint assays amplifying six fragments of different length of the HPRT1 gene and RNA Integrity Number (RIN). To evaluate the difference of tissue functionality among the stabilization methods tested, RT-qPCRs were performed focusing on the differential expression of the HPRT1, SNRPD3 and Jun genes. RNA from the samples preserved with the RNL or SF-OCT method showed better integrity compared to SF and FFPE, measured by PCR-endpoint and RT-qPCR assays. However, only statistically significant differences were observed between the RNA from FFPE and other stabilization methods when gene expression of HPRT1, SNRPD3 and Jun housekeeping genes were determined by RT-qPCR. For the three mentioned genes, Cq and RIN values were highly correlated. The present work describes the fragility of SF samples, being critical the moment just before RNA extraction, although further experiments of tissue RNA are needed. Standardization pre-analytic workflow can lead to improved reproducibility between biomedical research studies. The present study demonstrated clear evidences about the impact of the stabilization method on RNA derived from lung human tissue samples.
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spelling pubmed-70467792020-03-05 Impact of different stabilization methods on RT-qPCR results using human lung tissue samples Esteva-Socias, Margalida Gómez-Romano, Fernando Carrillo-Ávila, José Antonio Sánchez-Navarro, Alicia Loreto Villena, Cristina Sci Rep Article Aiming to increase the reproducibility of biomedical research results, biobanks obtain human tissues of the highest quality and carry out different storage methods adapted to the needs of analytical technique to be performed by the biomedical researchers. However, there is much controversy and little data concerning the real impact of different stabilization methods on tissue quality, integrity and functionality of derived biomolecules. The influence of four stabilization methods [RNAlater (RNL), snap freezing (SF), snap freezing using Optimal Cutting Tissue compound (SF-OCT) and formalin-fixed paraffin-embedded (FFPE)] on RNA quality and integrity was evaluated in paired samples of lung tissue. RNA integrity was evaluated through PCR-endpoint assays amplifying six fragments of different length of the HPRT1 gene and RNA Integrity Number (RIN). To evaluate the difference of tissue functionality among the stabilization methods tested, RT-qPCRs were performed focusing on the differential expression of the HPRT1, SNRPD3 and Jun genes. RNA from the samples preserved with the RNL or SF-OCT method showed better integrity compared to SF and FFPE, measured by PCR-endpoint and RT-qPCR assays. However, only statistically significant differences were observed between the RNA from FFPE and other stabilization methods when gene expression of HPRT1, SNRPD3 and Jun housekeeping genes were determined by RT-qPCR. For the three mentioned genes, Cq and RIN values were highly correlated. The present work describes the fragility of SF samples, being critical the moment just before RNA extraction, although further experiments of tissue RNA are needed. Standardization pre-analytic workflow can lead to improved reproducibility between biomedical research studies. The present study demonstrated clear evidences about the impact of the stabilization method on RNA derived from lung human tissue samples. Nature Publishing Group UK 2020-02-27 /pmc/articles/PMC7046779/ /pubmed/32108147 http://dx.doi.org/10.1038/s41598-020-60618-x Text en © The Author(s) 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Esteva-Socias, Margalida
Gómez-Romano, Fernando
Carrillo-Ávila, José Antonio
Sánchez-Navarro, Alicia Loreto
Villena, Cristina
Impact of different stabilization methods on RT-qPCR results using human lung tissue samples
title Impact of different stabilization methods on RT-qPCR results using human lung tissue samples
title_full Impact of different stabilization methods on RT-qPCR results using human lung tissue samples
title_fullStr Impact of different stabilization methods on RT-qPCR results using human lung tissue samples
title_full_unstemmed Impact of different stabilization methods on RT-qPCR results using human lung tissue samples
title_short Impact of different stabilization methods on RT-qPCR results using human lung tissue samples
title_sort impact of different stabilization methods on rt-qpcr results using human lung tissue samples
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7046779/
https://www.ncbi.nlm.nih.gov/pubmed/32108147
http://dx.doi.org/10.1038/s41598-020-60618-x
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