Cherchi Roberto, Cusano Roberto, Orrù Sandro, Ferrari Paolo A, Massidda Matteo, Fotia Giorgio, De Matteis Sara, Cocco Pierluigi
Operative Unit of Thoracic Surgery, Hospital G. Brotzu, Cagliari, Italy.
CRS4-NGS Core, POLARIS Research Park, Pula-Cagliari, Italy.
Epigenet Insights. 2023 Apr 2;16:25168657231160985. doi: 10.1177/25168657231160985. eCollection 2023.
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.
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.
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, = .026-<.001; HiSeq = 0.156-0.412, = .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.
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.
呼气末冷凝液(EBC)采样被认为是一种检测生物大分子(包括miRNA)的侵入性较小且经济高效的方法。为了探索其作为石棉暴露早期效应生物标志物的可行性,我们通过使用两种不同的测序平台比较EBC和血浆中的miRNA谱,对男性志愿者进行了初步测试。
六名男性志愿者参与了测试,他们均已退休,未接触过灰尘或烟雾。从200μL EBC样本和相同体积的血浆样本中提取RNA。样本等分试样在两个实验室中使用两种不同的测序平台进行处理:MiSeq Illumina平台和性能更强的HiSeq Illumina平台。
HiSeq3000测序平台鉴定出的经独特分子索引(UMI)验证的miRNA数量是MiSeq平台的两倍。在使用任一平台的5/6受试者中,EBC计数与血浆计数之间的Spearman相关系数均具有统计学意义(MiSeq = 0.128 - 0.508,P = 0.026 - <0.001;HiSeq = 0.156 - 0.412,P = 0.001 - <0.001)。组内相关系数证实了两种生物样本在6名参与者中的miRNA谱的一致性。使用Bland-Altman图探索EBC和血浆样本之间的一致性表明,使用HiSeq3000平台可显著提高EBC miRNA的检测率。
我们的初步研究证实,使用HiSeq测序平台时,EBC采样是检测健康受试者miRNA谱的一种合适的非侵入性方法。