An optimized method for high-quality RNA extraction from distinctive intrinsic laryngeal muscles in the rat model.
An optimized method for high-quality RNA extraction from distinctive intrinsic laryngeal muscles in the rat model.
复制标题
从大鼠模型中，从独特的内在喉部肌肉中提取高质量RNA的优化方法。
DOI：
10.1038/s41598-022-25643-y
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发表时间：
2022-12-15
影响因子：
4.6
通讯作者：
Pitman, Michael J.
中科院分区：
文献类型：
--
作者：
Kemfack, Angela M.;Hernandez-Morato, Ignacio;Moayedi, Yalda;Pitman, Michael J.
从死后组织中提取高质量RNA的挑战限制了RNA测序（RNA-seq）在某些骨骼肌群（包括喉内肌（ILMs））中的应用。本研究确定了导致从ILMs中提取不合格RNA的关键因素，并建立了一种适合的高通量分析方法。在这里，组织处理的标准技术进行了调整，并确定了在标本制备过程中控制混淆效应的有效手段。实验程序始终提供足够的完整总RNA (N = 68)， RIN范围在7.0到8.6之间，这是使用标准RNA纯化方案前所未有的。本研究通过在不同的产后时间点和不同的ilm进行重复试验，证实了工作流程的可重复性。90份RNA样品的高通量诊断显示，测序比对得分均低于70%，验证了提取策略。标准和实验条件之间的显着差异表明规避了挑战并广泛适用于其他骨骼肌。鉴于对声音、吞咽和气道疾病的治疗见解的前景，这项研究仍在进行中。目前的方法支持开创性的全球转录组研究在喉部以前没有根据的文献。
Challenges related to high-quality RNA extraction from post-mortem tissue have limited RNA-sequencing (RNA-seq) application in certain skeletal muscle groups, including the intrinsic laryngeal muscles (ILMs). The present study identified critical factors contributing to substandard RNA extraction from the ILMs and established a suitable method that permitted high-throughput analysis. Here, standard techniques for tissue processing were adapted, and an effective means to control confounding effects during specimen preparation was determined. The experimental procedure consistently provided sufficient intact total RNA (N = 68) and RIN ranging between 7.0 and 8.6, which was unprecedented using standard RNA purification protocols. This study confirmed the reproducibility of the workflow through repeated trials at different postnatal time points and across the distinctive ILMs. High-throughput diagnostics from 90 RNA samples indicated no sequencing alignment scores below 70%, validating the extraction strategy. Significant differences between the standard and experimental conditions suggest circumvented challenges and broad applicability to other skeletal muscles. This investigation remains ongoing given the prospect of therapeutic insights to voice, swallowing, and airway disorders. The present methodology supports pioneering global transcriptome investigations in the larynx previously unfounded in literature.
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