LoCoco Peter M, Boyd Jacob T, Espitia Olaya Claudia M, Furr Ashley R, Garcia Dawn K, Weldon Korri S, Zou Yi, Locke Erin, Tobon Alejandro, Lai Zhao, Ruparel Shivani B, Ruparel Nikita B, Hargreaves Kenneth M
Department of Endodontics, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA.
University of Arizona Cancer Center, Tucson, AZ, USA.
Pain Rep. 2020 Mar 27;5(2):e818. doi: 10.1097/PR9.0000000000000818. eCollection 2020 Mar-Apr.
Comprehensive mRNA sequencing is a powerful tool for conducting unbiased, quantitative differential gene expression analysis. However, the reliability of these data is contingent on the extraction of high-quality RNA from samples. Preserving RNA integrity during extraction can be problematic, especially in tissues such as skin with dense, connective matrices and elevated ribonuclease expression. This is a major barrier to understanding the influences of altered gene expression in many preclinical pain models and clinical pain disorders where skin is the site of tissue injury.
This study developed and evaluated extraction protocols for skin and other tissues to maximize recovery of high-integrity RNA needed for quantitative mRNA sequencing.
Rodent and human tissue samples underwent one of the several different protocols that combined either RNA-stabilizing solution or snap-freezing with bead milling or cryosectioning. Indices of RNA integrity and purity were assessed for all samples.
Extraction of high-integrity RNA is highly dependent on the methods used. Bead-milling skin collected in RNA-stabilizing solution resulted in extensive RNA degradation. Snap-freezing in liquid nitrogen was required for skin and highly preferable for other tissues. Skin also required cryosectioning to achieve effective penetration of RNA-stabilizing solution to preserve RNA integrity, whereas bead milling could be used instead with other tissues. Each method was reproducible across multiple experimenters. Electrophoretic anomalies that skewed RNA integrity value assignment required manual correction and often resulted in score reduction.
To achieve the potential of quantitative differential gene expression analysis requires verification of tissue-dependent extraction methods that yield high-integrity RNA.
全面的mRNA测序是进行无偏倚、定量差异基因表达分析的强大工具。然而，这些数据的可靠性取决于从样本中提取高质量RNA。在提取过程中保持RNA完整性可能存在问题，尤其是在皮肤等组织中，其具有致密的结缔组织基质且核糖核酸酶表达升高。这是理解许多临床前疼痛模型和临床疼痛障碍中基因表达改变影响的主要障碍，在这些模型和障碍中皮肤是组织损伤的部位。
本研究开发并评估了用于皮肤和其他组织的提取方案，以最大限度地回收定量mRNA测序所需的高完整性RNA。
啮齿动物和人类组织样本采用几种不同方案之一进行处理，这些方案将RNA稳定溶液或速冻与珠磨或冷冻切片相结合。评估所有样本的RNA完整性和纯度指标。
高完整性RNA的提取高度依赖于所使用的方法。在RNA稳定溶液中收集的皮肤进行珠磨会导致大量RNA降解。皮肤需要在液氮中速冻，对其他组织则更可取。皮肤还需要冷冻切片，以使RNA稳定溶液有效渗透以保持RNA完整性，而其他组织可以使用珠磨代替。每种方法在多个实验者之间均可重复。导致RNA完整性值分配偏差的电泳异常需要人工校正，且常常导致分数降低。
要实现定量差异基因表达分析的潜力，需要验证能产生高完整性RNA的组织依赖性提取方法。
