Next Generation Sequencing for miRNA Detection on the Exhaled Breath Condensate: A Pilot Study.
Author(s): Cherchi R, Cusano R, Orrù S, Ferrari PA, Massidda M, Fotia G, De Matteis S, Cocco P
Publication: Epigenet Insights, 2023, Vol. 16, Page 25168657231160985
PubMed ID: 37025420 PubMed Review Paper? No
Purpose of Paper
This paper compared microRNA (miRNA, miR) detection and counts in case-matched exhaled breath condensate (EBC) and plasma from six healthy volunteers using the MiSeq and HiSeq next generation sequencing instruments.
Conclusion of Paper
As expected, due to increased sensitivity, close to twice as many miRNA reads were generated using the HiSeq than the MiSeq instrument (600 versus 303). The number of detected miRNA reads was not correlated between case-matched EBC and plasma from any of the six volunteers regardless of platform. However, miRNA counts were significantly correlated between EBC and plasma specimens from four of six volunteers using MiSeq and HiSeq instruments. Bland-Altman plots revealed there were a greater number of miRNA counts in plasma than EBC with the MiSeq instrument, but the same was not true when the HiSeq instrument was used for sequencing. The authors stated that this was indicative that MiSeq was not sensitive enough for analysis of EBC specimens. While only ten miRNAs were detected in EBC specimens collected from all six volunteers using the MiSeq instrument, 239 miRNAs were detected in EBC specimens from of all six volunteers using the HiSeq instrument. The authors conclude that using HiSeq rather than MiSeq improved miENA detection in EBC.
Studies
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Study Purpose
This study compared miRNA detection and counts in case-matched exhaled breath condensate (EBC) and plasma specimens collected from six healthy volunteers using the MiSeq and HiSeq NGS instruments. EBC was collected from six healthy male volunteers that exhaled in a Volmet 20 device for 15-20 minutes to achieve a standardized total volume of 100 L; the exhaled breath was then chilled to -5°C with a TURBO-DECCS System for the collection of the liquid condensate specimen. A total of 1-2 mL of EBC was collected from each participant. From each volunteer, EDTA plasma was separated from blood by centrifugation at 3000 g at 4°C for 10 min followed by centrifugation at 14,000 g (duration not specified) and then frozen at -80°C. RNA was extracted from EBC and plasma using the miRNeasy Serum/Plasma Advanced Kit. Sequencing libraries were prepared with the QIAseq miRNA Library Kit, quantified by Qubit, and analyzed for fragment length with a Bioanalyer. Libraries were sequenced on a MiSeq and a HiSeq Instrument using independently prepared libraries.
Summary of Findings:
As expected, due to increased sensitivity, close to twice as many miRNA reads were generated using the HiSeq than the MiSeq instrument (600 versus 303). The number of detected miRNA reads was not correlated between case-matched EBC and plasma from any of the six volunteers regardless of platform. However, miRNA counts were significantly correlated between EBC and plasma specimens from four of six volunteers using MiSeq (ρ=0.325-0.508, P<0.0001, all) and HiSeq instruments MiSeq (ρ=0.169-0.412, P<0.0001) with significantly correlated levels observed using both platforms in specimens from three of the six volunteers. Bland-Altman plots revealed there were a greater number of miRNA counts in plasma than EBC with the MiSeq instrument, but the same was not true when the HiSeq instrument was used for sequencing. The authors stated that this was indicative that MiSeq was not sensitive enough for analysis of EBC specimens. While only ten miRNAs were detected in EBC specimens collected from all six volunteers using the MiSeq instrument, 239 miRNAs were detected in EBC specimens from of all six volunteers using the HiSeq instrument. The authors conclude that using HiSeq rather than MiSeq improved miENA detection in EBC.
Biospecimens
Preservative Types
- Frozen
- None (Fresh)
Diagnoses:
- Normal
Platform:
Analyte Technology Platform RNA Next generation sequencing Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Next generation sequencing Specific Technology platform HiSeq
MiSeq
Biospecimen Acquisition Biospecimen location Plasma
Exhaled breath condensate