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

Area of Science:

• Pulmonary Medicine
• Molecular Biology
• Genomics

Background:

• Assessing airway inflammation in COPD typically requires invasive methods and complex sample handling.
• Current methods for sputum analysis are labor-intensive and require specialized expertise.
• There is a need for simpler, non-invasive techniques to study COPD pathogenesis.

Purpose of the Study:

• To develop and validate a novel non-invasive, processing-free method for RNA sequencing (RNAseq) of spontaneous sputum.
• To assess airway inflammation in chronic obstructive pulmonary disease (COPD) patients.
• To determine if spontaneous sputum RNAseq can accurately reflect COPD endotypes.

Main Methods:

• Spontaneous sputum samples were collected and stabilized using PrimeStore® Molecular Transport Medium.
• RNA was isolated using an optimized RNAzol® method after 8 days of ambient temperature storage.
• RNA integrity (DV200 >30%) and yield (>50 ng) were assessed before RNAseq; results were validated using induced sputum samples and ssGSEA.

Main Results:

• Stabilized spontaneous sputum yielded RNA with DV200 >30% and 70% of samples met the yield requirement (>50 ng).
• Gene expression analysis showed high correlation between samples processed with or without plug selection.
• Single-sample gene-set enrichment analysis (ssGSEA) of spontaneous sputum revealed pathways related to COPD inflammation, immune response, and oxidative stress, with 70% overlap with induced sputum results.

Conclusions:

• The developed method is a non-invasive, processing-free approach for RNAseq analysis of spontaneous sputum.
• This technique accurately represents the COPD endotype by reflecting sputum cell inflammation.
• The findings support using spontaneous sputum RNAseq as a viable tool for COPD research and potentially clinical assessment.