从钙化人心脏瓣膜中提取总RNA用于基因表达分析。
Extraction of Total RNA from Calcified Human Heart Valves for Gene Expression Analysis.
作者信息
Cecília Valadares Ana, Gorki Hagen, Liebold Andreas, Hoenicka Markus
机构信息
University of Ulm Medical Center, Department of Cardiothoracic and Vascular Surgery, Ulm, Germany. Electronic correspondence:
University of Ulm Medical Center, Department of Cardiothoracic and Vascular Surgery, Ulm, Germany.
出版信息
J Heart Valve Dis. 2017 Mar;26(2):185-192.
BACKGROUND
The isolation of high-quality RNA is an important first step in gene expression studies. However, difficult tissue disruption, low cell content and low RNA content makes consistent RNA extraction from human aortic valve tissue a challenging task.
METHODS
A protocol has been developed for the successful isolation of high-quality RNA from human aortic valve samples by optimizing RNA extraction protocols based on a comparison of commercial kits.
RESULTS
Guanidinium thiocyanate-phenolchloroform extraction was found to be a prerequisite for successful purification. Two protocols based on this extraction were further optimized. RNA quality and quantity were assessed spectrophotometrically, using a Bioanalyzer and by PCR analysis of several housekeeping genes. Optimized parameters included storage in RNAlater™, DNase digestion, the amount of tissue, homogenization time, and freezing of tissue after homogenization.
CONCLUSIONS
The modified protocol for fatty and fibrous tissue achieved satisfactory results for gene expression analysis of human aortic valve samples.
背景
高质量RNA的分离是基因表达研究中重要的第一步。然而，人主动脉瓣组织难以破坏、细胞含量低以及RNA含量低，使得从该组织中持续提取RNA成为一项具有挑战性的任务。
方法
通过基于商用试剂盒比较优化RNA提取方案，已开发出一种从人主动脉瓣样本中成功分离高质量RNA的方案。
结果
发现异硫氰酸胍 - 苯酚氯仿提取是成功纯化的先决条件。基于此提取方法的两种方案进一步优化。使用分光光度计、生物分析仪并通过对几个管家基因的PCR分析来评估RNA的质量和数量。优化参数包括储存在RNAlater™中、DNase消化、组织量、匀浆时间以及匀浆后组织的冷冻。
结论
针对脂肪和纤维组织的改良方案在人主动脉瓣样本的基因表达分析中取得了令人满意的结果。
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