Maiocchi Sophie, Collins Elizabeth N, Peterson Andrew R, Alexander Kyle C, McGlamery Dalton J, Cassidy Noah A, Ikonomidis John S, Akerman Adam W
Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7545, USA.
Department of Surgery, Division of Cardiothoracic Surgery, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7065, USA.
Vessel Plus. 2023;7. doi: 10.20517/2574-1209.2023.69. Epub 2023 Nov 15.
MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate translation and are involved in many pathological processes. They have emerged as promising biomarkers for diagnosis of conditions such as aortic aneurysm disease. Quantifying miRNAs in plasma is uniquely challenging because of the lack of standardized reproducible protocols. To facilitate the independent verification of conclusions, it is necessary to provide a thorough disclosure of all pertinent experimental details. In this technical note, we present a comprehensive protocol for quantifying plasma miRNAs using droplet digital PCR. We detail the entire workflow, including blood collection, plasma processing, cryo-storage, miRNA isolation, reverse transcription, droplet generation, PCR amplification, fluorescence reading, and data analysis. We offer comprehensive guidance regarding optimization, assay conditions, expected results, and insight into the troubleshooting of common issues. The stepwise normalization and detailed methodological guide enhance reproducibility. Moreover, multiple portions of this protocol may be automated. The data provided in this technical note is demonstrative of the values typically obtained when following its steps. To facilitate standardization in data reporting, we include a table of expected aortic aneurysm-related miRNA levels in healthy human plasma. This versatile protocol can be easily adapted to quantify most circulating miRNAs in plasma, making it a valuable resource for diagnostic development.
微小RNA（miRNA）是一类小型非编码RNA分子，可调节翻译过程，并参与许多病理过程。它们已成为诸如主动脉瘤疾病等病症诊断中很有前景的生物标志物。由于缺乏标准化的可重复方案，对血浆中的miRNA进行定量具有独特的挑战性。为便于对结论进行独立验证，有必要全面披露所有相关实验细节。在本技术说明中，我们介绍了一种使用液滴数字PCR定量血浆miRNA的综合方案。我们详细说明了整个工作流程，包括血液采集、血浆处理、冷冻保存、miRNA分离、逆转录、液滴生成、PCR扩增、荧光读取和数据分析。我们提供了关于优化、检测条件、预期结果的全面指导，以及对常见问题故障排除的见解。逐步归一化和详细的方法指南提高了可重复性。此外，该方案的多个部分可以自动化。本技术说明中提供的数据证明了遵循其步骤时通常获得的值。为便于数据报告的标准化，我们列出了健康人血浆中与主动脉瘤相关的miRNA水平预期表。这个通用方案可以很容易地适用于定量血浆中大多数循环miRNA，使其成为诊断开发的宝贵资源。
