An optimized method for high-quality RNA extraction from distinctive intrinsic laryngeal muscles in the rat model

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.从死后组织中提取高质量RNA所面临的诸多挑战，限制了RNA测序（RNA-seq）在部分骨骼肌群中的应用，其中包括喉内肌（ILMs）。本研究明确了导致喉内肌RNA提取质量不达标的关键因素，并建立了可用于高通量分析的适宜方法。本研究对标准组织处理技术进行了改良，并确定了一种可有效控制样本制备过程中混杂效应的方案。该实验流程可稳定获得足量完整总RNA（N=68），且RNA完整性数（RIN）介于7.0至8.6之间，这一结果在采用标准RNA纯化方案的情况下此前从未实现。本研究通过在不同出生后时间节点以及各类喉内肌中开展重复实验，验证了该实验流程的可重复性。对90份RNA样本进行的高通量检测结果显示，所有测序比对评分均不低于70%，证实了该提取策略的有效性。标准方案与实验方案之间存在的显著差异表明，本研究成功克服了相关难题，且该方法可广泛应用于其他骨骼肌研究。鉴于该研究有望为声带、吞咽及气道疾病提供治疗相关的新见解，本调查仍在持续进行中。本研究建立的方法可为此前尚无文献报道的喉部全局转录组研究提供支撑，助力该领域的开创性探索。