Abstract
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 (DV200) >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 DV200 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.
Highlights
Sputum induction and its subsequent processing has become the gold standard clinical tool for accessing airway inflammation in chronic obstructive pulmonary disease (COPD) (Lacy et al, 2005; Dragonieri et al, 2009)
To assess if the transcriptome generated from a specific collection and handling method would reflect a true endotype of COPD inflammation, the outcome of single sample gene set enrichment analysis (ssGSEA) was validated against the transcriptome of an independent set of processed induced sputum samples from COPD patients
® Following RNAzol extraction only spontaneous sputum samples stabilized in MTM generated RNA with an average DV200 > 30%
Summary
Sputum induction and its subsequent processing has become the gold standard clinical tool for accessing airway inflammation in chronic obstructive pulmonary disease (COPD) (Lacy et al, 2005; Dragonieri et al, 2009). The technique is generally well-tolerated and safe (Bathoorn et al, 2007; Tangedal et al, 2014) but remains a semiinvasive procedure (Koutsokera et al, 2013) that may create discomfort in COPD patients (Taube et al, 2001; Makris et al, 2006). 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
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