Next Generation Sequencing for miRNA Detection on the Exhaled Breath Condensate: A Pilot Study

Roberto Cherchi; Roberto Cusano; Sandro Orrù; Paolo A Ferrari; Matteo Massidda; Giorgio Fotia; Sara De Matteis; Pierluigi Cocco

doi:10.1177/25168657231160985

Next Generation Sequencing for miRNA Detection on the Exhaled Breath Condensate: A Pilot Study

Epigenet Insights. 2023 Apr 2:16:25168657231160985. doi: 10.1177/25168657231160985. eCollection 2023.

Authors

Roberto Cherchi¹, Roberto Cusano², Sandro Orrù^{3

4}, Paolo A Ferrari¹, Matteo Massidda², Giorgio Fotia², Sara De Matteis⁴, Pierluigi Cocco⁵

Affiliations

¹ Operative Unit of Thoracic Surgery, Hospital G. Brotzu, Cagliari, Italy.
² CRS4-NGS Core, POLARIS Research Park, Pula-Cagliari, Italy.
³ Operative Unit of Medical Genetics, Health Agency of Sardinia, Hospital Binaghi, Cagliari, Italy.
⁴ Department of Medical Sciences and Public Health, University of Cagliari, Monserrato-Cagliari, Italy.
⁵ Centre for Occupational and Environmental Health, Division of Population Health, Health Services Research & Primary Care, University of Manchester, Manchester, UK.

Abstract

Introduction: Exhaled breath condensate (EBC) sampling has been suggested as a less-invasive and cost-effective method to detect biological macromolecules, including miRNA. To explore the feasibility of its use as a biomarker of early effects of asbestos exposure, we conducted a preliminary test on male volunteers by comparing the miRNA profile in the EBC and the plasma using 2 different sequencing platforms.

Methods: Six male volunteers, all retired and unexposed to dust or fumes, participated in the test. RNA was extracted from 200 μL EBC samples and same-size plasma samples. Sample aliquots were processed in 2 laboratories using 2 different sequencing platforms: a MiSeq Illumina^® platform and a more performing HiSeq Illumina^® platform.

Results: The HiSeq3000^® sequencing platform identified twice as many unique molecular indexes (UMI)-validated miRNA as the MiSeq^® platform. The Spearman's correlation coefficient between EBC counts and plasma counts was significant in 5/6 subjects with either platform (MiSeq^® = 0.128-0.508, P = .026-<.001; HiSeq^® = 0.156-0.412, P = .001-<.001). The intraclass correlation coefficient confirmed the consistency of the miRNA profile over the 6 participants with both biospecimens. Exploring the agreement between the EBC and plasma samples with Bland-Altman plots showed that using the HiSeq3000^® platform substantially improved the EBC miRNA detection rate.

Conclusion: Our preliminary study confirms that, when using the HiSeq^® sequencing platform, EBC sampling is a suitable, non-invasive method to detect the miRNA profile in healthy subjects.

Keywords: biomarkers; exhaled breath condensate; miRNA; next generation sequencing.