Feasibility of Small RNA Profiling by Next-Generation Sequencing in Exhaled Breath Condensate and Its Modulation by Maximal Exercise: a Pilot Study

Background: Exhaled breath condensate (EBC) is a non-invasively collected biofluid containing volatile and non-volatile compounds, including small RNAs. Small RNA profiling in EBC appears to be a promising approach for identifying and developing minimally invasive biomarkers for lung diseases; however, methodological evidence remains limited and heterogeneous.

Objectives: To evaluate the technical feasibility of small RNA extraction and next-generation sequencing (NGS) from EBC and to characterise small RNA profiles at rest and after maximal exercise, using EBC as a model matrix for dynamic physiological changes.

Methods: In a pilot study conducted as part of statutory research at the Medical University of Białystok (B.SUB.25.512 and B.SUB.25.529), we analysed 22 EBC samples collected with the RTube device from 11 healthy volunteers and 3 patients with lung cancer and co-existing COPD (GOLD 3-4), at rest and after standardised maximal exercise confirmed by cardiopulmonary exercise testing (CPET). Total RNA was extracted using a commercial high-throughput RNA isolation kit designed for biological samples with low nucleic acid content, the miRNeasy Serum/Plasma Advanced Kit (Qiagen), according to the manufacturer's protocol with modifications. Small RNA libraries were generated using the high-sensitivity, low-input TrueQuant Small RNA-Seq Library Preparation Kit (GenXPro GmbH) with Unique Molecular Identifiers (UMIs) and sequenced on an Illumina platform. Reads were processed with Cutadapt and FastQC, mapped iteratively to the hg38 (Homo sapiens) reference genome using Bowtie2, aligned against an array of RNA databases, including miRNA (miRBase), tRNA (GtRNAdb), piRNA (piRNAdb), ncRNA (ENSEMBL), and cDNA (ENSEMBL) and quantified with HTSeq. Differential expression analysis (DEA) and principal component analysis (PCA) were performed with DESeq2.