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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