Reliable approaches to extract high-integrity RNA from skin and other pertinent tissues used in pain research
Reliable approaches to extract high-integrity RNA from skin and other pertinent tissues used in pain research
复制标题
DOI：
10.1097/pr9.0000000000000818
复制
发表时间：
2020-03-01
期刊：
影响因子：
4.8
通讯作者：
Hargreaves, Kenneth M.
中科院分区：
文献类型：
--
作者：
LoCoco, Peter M.;Boyd, Jacob T.;Hargreaves, Kenneth M.
简介：全面的mRNA测序是进行无偏、定量差异基因表达分析的有力工具。然而，这些数据的可靠性取决于从样品中提取高质量的RNA。在提取过程中保持RNA的完整性可能是有问题的，特别是在具有致密结缔基质和核糖核酸酶表达升高的组织如皮肤中。这是理解在许多临床前疼痛模型和临床疼痛疾病（其中皮肤是组织损伤部位）中基因表达改变的影响的主要障碍。目的：本研究开发并评估了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品经历了几种不同的方案之一，这些方案将RNA稳定溶液或快速冷冻与珠磨或冷冻切片相结合。RNA的完整性和纯度的指数进行了评估为所有samples.Results：高完整性RNA的提取是高度依赖于所使用的方法。在RNA稳定溶液中收集的珠磨皮肤导致广泛的RNA降解。皮肤需要在液氮中快速冷冻，其他组织则非常优选。皮肤也需要冷冻切片，以实现RNA稳定溶液的有效渗透，从而保持RNA的完整性，而珠磨可用于其他组织。每种方法在多个实验者中是可重复的。导致RNA完整性值分配偏差的电泳异常需要手动纠正，并且通常会导致评分降低。结论：为了实现定量差异基因表达分析的潜力，需要验证可产生高完整性RNA的组织依赖性提取方法。
Introduction: 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.Objective: This study developed and evaluated extraction protocols for skin and other tissues to maximize recovery of high-integrity RNA needed for quantitative mRNA sequencing.Methods: 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.Results: 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.Conclusion: To achieve the potential of quantitative differential gene expression analysis requires verification of tissue-dependent extraction methods that yield high-integrity RNA.