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A Novel Processing-Free Method for RNAseq Analysis of Spontaneous Sputum in Chronic Obstructive Pulmonary Disease

Background: Assessments of airways inflammation in patients with chronic obstructive pulmonary disease (COPD) require semi-invasive procedures and specialized sample processing know-how. In this study we aimed to set up and validate a novel non-invasive processing-free method for RNA sequencing (RNA...

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Autores principales: Ricci, Francesca, Bassi, Michele, McGeough, Cathy M., Jellema, Gera L., Govoni, Mirco
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8417251/
https://www.ncbi.nlm.nih.gov/pubmed/34489698
http://dx.doi.org/10.3389/fphar.2021.704969
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author Ricci, Francesca
Bassi, Michele
McGeough, Cathy M.
Jellema, Gera L.
Govoni, Mirco
author_facet Ricci, Francesca
Bassi, Michele
McGeough, Cathy M.
Jellema, Gera L.
Govoni, Mirco
author_sort Ricci, Francesca
collection PubMed
description Background: Assessments of airways inflammation in patients with chronic obstructive pulmonary disease (COPD) require semi-invasive procedures and specialized sample processing know-how. In this study we aimed to set up and validate a novel non-invasive processing-free method for RNA sequencing (RNAseq) of spontaneous sputum samples collected from COPD patients. Methods: Spontaneous sputum samples were collected and stabilized, with or without selection of plugs and with or without the use of a stabilizer specifically formulated for downstream diagnostic testing (PrimeStore® Molecular Transport Medium). After 8 days storage at ambient temperature RNA was isolated according to an optimized RNAzol® method. An average percentage of fragments longer than 200 nucleotides (DV(200)) >30% and an individual yield >50 ng were required for progression of samples to sequencing. Finally, to assess if the transcriptome generated would reflect a true endotype of COPD inflammation, the outcome of single-sample gene-set enrichment analysis (ssGSEA) was validated using an independent set of processed induced sputum samples. Results: RNA extracted from spontaneous sputum using a stabilizer showed an average DV(200) higher than 30%. 70% of the samples had a yield >50 ng and were submitted to downstream analysis. There was a straightforward correlation in terms of gene expression between samples handled with or without separation of plugs. This was also confirmed by principal component analysis and ssGSEA. The top ten enriched pathways resulting from spontaneous sputum ssGSEA were associated to features of COPD, namely, inflammation, immune responses and oxidative stress; up to 70% of these were in common within the top ten enriched pathways resulting from induced sputum ssGSEA. Conclusion: This analysis confirmed that the typical COPD endotype was represented within spontaneous sputum and supported the current method as a non-invasive processing-free procedure to assess the level of sputum cell inflammation in COPD patients by RNAseq analysis.
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spelling pubmed-84172512021-09-05 A Novel Processing-Free Method for RNAseq Analysis of Spontaneous Sputum in Chronic Obstructive Pulmonary Disease Ricci, Francesca Bassi, Michele McGeough, Cathy M. Jellema, Gera L. Govoni, Mirco Front Pharmacol Pharmacology Background: Assessments of airways inflammation in patients with chronic obstructive pulmonary disease (COPD) require semi-invasive procedures and specialized sample processing know-how. In this study we aimed to set up and validate a novel non-invasive processing-free method for RNA sequencing (RNAseq) of spontaneous sputum samples collected from COPD patients. Methods: Spontaneous sputum samples were collected and stabilized, with or without selection of plugs and with or without the use of a stabilizer specifically formulated for downstream diagnostic testing (PrimeStore® Molecular Transport Medium). After 8 days storage at ambient temperature RNA was isolated according to an optimized RNAzol® method. An average percentage of fragments longer than 200 nucleotides (DV(200)) >30% and an individual yield >50 ng were required for progression of samples to sequencing. Finally, to assess if the transcriptome generated would reflect a true endotype of COPD inflammation, the outcome of single-sample gene-set enrichment analysis (ssGSEA) was validated using an independent set of processed induced sputum samples. Results: RNA extracted from spontaneous sputum using a stabilizer showed an average DV(200) higher than 30%. 70% of the samples had a yield >50 ng and were submitted to downstream analysis. There was a straightforward correlation in terms of gene expression between samples handled with or without separation of plugs. This was also confirmed by principal component analysis and ssGSEA. The top ten enriched pathways resulting from spontaneous sputum ssGSEA were associated to features of COPD, namely, inflammation, immune responses and oxidative stress; up to 70% of these were in common within the top ten enriched pathways resulting from induced sputum ssGSEA. Conclusion: This analysis confirmed that the typical COPD endotype was represented within spontaneous sputum and supported the current method as a non-invasive processing-free procedure to assess the level of sputum cell inflammation in COPD patients by RNAseq analysis. Frontiers Media S.A. 2021-08-19 /pmc/articles/PMC8417251/ /pubmed/34489698 http://dx.doi.org/10.3389/fphar.2021.704969 Text en Copyright © 2021 Ricci, Bassi, McGeough, Jellema and Govoni. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Pharmacology
Ricci, Francesca
Bassi, Michele
McGeough, Cathy M.
Jellema, Gera L.
Govoni, Mirco
A Novel Processing-Free Method for RNAseq Analysis of Spontaneous Sputum in Chronic Obstructive Pulmonary Disease
title A Novel Processing-Free Method for RNAseq Analysis of Spontaneous Sputum in Chronic Obstructive Pulmonary Disease
title_full A Novel Processing-Free Method for RNAseq Analysis of Spontaneous Sputum in Chronic Obstructive Pulmonary Disease
title_fullStr A Novel Processing-Free Method for RNAseq Analysis of Spontaneous Sputum in Chronic Obstructive Pulmonary Disease
title_full_unstemmed A Novel Processing-Free Method for RNAseq Analysis of Spontaneous Sputum in Chronic Obstructive Pulmonary Disease
title_short A Novel Processing-Free Method for RNAseq Analysis of Spontaneous Sputum in Chronic Obstructive Pulmonary Disease
title_sort novel processing-free method for rnaseq analysis of spontaneous sputum in chronic obstructive pulmonary disease
topic Pharmacology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8417251/
https://www.ncbi.nlm.nih.gov/pubmed/34489698
http://dx.doi.org/10.3389/fphar.2021.704969
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