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用于检测呼出气冷凝物中微小RNA的下一代测序:一项初步研究。

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

作者信息

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

机构信息

Operative Unit of Thoracic Surgery, Hospital G. Brotzu, Cagliari, Italy.

CRS4-NGS Core, POLARIS Research Park, Pula-Cagliari, Italy.

出版信息

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

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,  = .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.

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.

摘要

引言

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。

方法

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。

结果

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。

结论

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。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6187/10070752/717e7fe9cb92/10.1177_25168657231160985-fig1.jpg

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