Next Generation Sequencing for miRNA Detection on the Exhaled Breath Condensate: A Pilot Study
Roberto Maria Cherchi, Roberto Cusano, Sandro Orrù, Paolo Albino Ferrari, Matteo Massidda, Giorgio Fotia, Sara De Matteis, Pierluigi Cocco
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.