Small RNA Profiling in EBC in Athletes (AirBiopsy)

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.

Study Overview

• Status: Completed
• Conditions: Athlete; Exhaled Breath Condensate; Cardio-pulmonary Function; RNA Profile

• Study Type: Observational
• Enrollment (Actual): 25

Contacts and Locations

Study Locations

• Poland
  • Podlaskie Voivodeship
    • Bialystok, Podlaskie Voivodeship, Poland, 15-540
      • Department of Respiratory Physiopathology, Medical University of Bialystok

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Adult; Older Adult
• Accepts Healthy Volunteers: Yes

• Sampling Method: Non-Probability Sample

Study Population

healthy volouteers, amateur athletes (runners) and lung cancer patients with stage IIIb or VI disease

Description

Inclusion Criteria:

• ability to perform CPET
• ability to perform spirometry

Exclusion Criteria:

• lack of informed consent
• absolute contradictions to CPET or spirometry

Study Plan

How is the study designed?

Number of groups / cohorts

1

Cohorts and Interventions

Group / Cohort
Exhaled breath condensate donors; athletes (22) and lung cancer (3) donors of EBC

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Time Frame
smallRNA profile	From August 2025 to December 2025
EBC collection	From January 2025 to August 2025

Collaborators and Investigators

• Sponsor: Medical University of Bialystok

Study record dates

Study Major Dates

• Study Start (Actual): January 5, 2025
• Primary Completion (Actual): August 30, 2025
• Study Completion (Actual): December 15, 2025

Study Registration Dates

• First Submitted: December 23, 2025
• First Submitted That Met QC Criteria: December 23, 2025
• First Posted (Actual): January 7, 2026

Study Record Updates

• Last Update Posted (Actual): February 6, 2026
• Last Update Submitted That Met QC Criteria: February 4, 2026
• Last Verified: February 1, 2026

More Information

Terms related to this study

• Other Study ID Numbers: APK.002.584.2024; B.SUB.25.512 (Other Identifier: Medical University of Bialystok)

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: YES
• IPD Plan Description: detailed RNA profile and clinical data available on demand
• IPD Sharing Time Frame: January 2026 to January 2027
• IPD Sharing Access Criteria: researchers and interested parties after an assessment of the data usage description and appropriate bioethical agreement
• IPD Sharing Supporting Information Type: STUDY_PROTOCOL; SAP; ICF; CSR

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No