Impact of fixative on recovery of mRNA from paraffin-embedded tissue
Impact of fixative on recovery of mRNA from paraffin-embedded tissue
复制标题
DOI：
10.1097/00019606-200406000-00008
复制
发表时间：
2004-06-01
影响因子：
--
通讯作者：
Heiskala, M
中科院分区：
文献类型：
--
作者：
Benchekroun, M;DeGraw, J;Heiskala, M
由于先进的组织分析工具的发展，如蛋白质组学和功能和结构基因组学，处理和保存样本的需求正在发生变化。对于基因表达分析，测试材料中必须存在完整和可提取的信使RNA。为了找到一种用于此类分析的最佳组织固定剂，我们评估了两种沉淀组织固定剂甲醇-丙酮和Carnoy‘s的形态、蛋白质抗原和RNA保持能力。这两种固定剂都保留了组织的形态和蛋白质表位，并可以提取质量明显高于福尔马林固定组织的总RNA。Carnoy的固定剂在保持RNA完整性方面比甲醇-丙酮表现得更好，特别是当固定的石蜡包埋组织块在室温下储存3个月以上时。从Carnoy‘s固定组织样本显微解剖的上皮细胞中提取的总RNA包含高达977个碱基对(BP)的P-肌动蛋白扩增子完整模板。由于基因表达分析在研究中和不久的将来在临床工作中的新作用，保存RNA的固定剂应该会取代福尔马林作为主要的人体组织固定剂。根据我们的数据，Carnoy的固定剂是一种新的人体组织样本主要固定剂的极佳候选者。
Due to the evolution of advanced tissue-analysis tools, such as proteomics and functional and structural genomics, the demands for handling and preserving samples are changing. For gene expression analysis, the presence of intact and extractable messenger RNA in the test material is mandatory. To find an optimal fixative for tissues aimed for such analyses, we evaluated the morphology-, protein antigen-, and RNA-maintaining abilities of 2 precipitating tissue fixatives, methanol-acetone and Carnoy's. Both fixatives preserved the morphology and protein epitopes of tissues and allowed extraction of total RNA that was of significantly higher quality than RNA extracted from formalin-fixed tissue. Carnoy's fixative performed better than methanol-acetone in maintaining the integrity of RNA, especially when the fixed, paraffin-embedded tissue blocks were stored at room temperature for more than 3 months. Total RNA extracted from epithelial cells microdissected from Carnoy's-fixed tissue samples contained intact template for up to a 977-base pair (bp) amplicon for P-actin. Because of the emerging role of gene expression analyses in research, and in clinical work in the near future, an RNA-preserving fixative should replace formalin as the primary human tissue fixative. According to our data, Carnoy's fixative is an excellent candidate for a new primary fixing reagent for human tissue samples.