Immuno-LCM: Laser capture microdissection of immunostained frozen sections for mRNA analysis
Immuno-LCM: Laser capture microdissection of immunostained frozen sections for mRNA analysis
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DOI：
10.1016/s0002-9440(10)65251-0
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发表时间：
1999-01-01
影响因子：
6
通讯作者：
Raffeld, M
中科院分区：
文献类型：
--
作者：
Fend, F;Emmert-Buck, MR;Raffeld, M
常规染色或未染色冰冻切片的显微解剖已被成功地用于获得纯化的细胞群体，用于分析具有复杂细胞类型混合的原始组织中的细胞特异性基因表达模式。然而，显微解剖的精确度和有效性往往受到仅通过形态识别不同细胞类型和结构的困难的限制。因此，我们开发了一种快速免疫染色程序，用于冰冻切片，然后进行激光捕获显微切割(LCM)和RNA提取，从而可以对免疫表型定义的细胞群体进行有针对性的mRNA分析。固定后，冰冻切片在无核糖核酸酶的条件下，使用快速三步链霉亲和素-生物素技术进行免疫染色，脱水并立即进行LCM。RNA是从捕获的组织中提取出来的，DNase I处理过，然后反转录。丙酮、甲醇或乙醇/丙酮固定切片在总处理时间12至25分钟后可提供出色的免疫染色。由于改进了对所需(或不所需)细胞类型的识别，显微切割的特异性、精确度和速度显著提高。从免疫染色组织中回收的mRNA质量较高。单步聚合酶链式反应能够从不到500个免疫染色的显微解剖细胞中扩增出管家基因(如β-肌动蛋白)和细胞特异性信息(如CD4或CD19)中超过600个碱基的片段。免疫LCM可以根据免疫表型或功能相关抗原的表达对分离出的细胞群体进行特定的mRNA分析，从而极大地扩展了我们研究异质组织中基因表达的能力。
Microdissection of routinely stained or unstained frozen sections has been used successfully to obtain purified cell populations for the analysis of cell-specific gene expression patterns in primary tissues with a complex mixture of cell types. However, the precision and usefulness of microdissection is frequently limited by the difficulty to identify different cell types and structures by morphology alone. We therefore developed a rapid immunostaining procedure for frozen sections followed by laser capture microdissection (LCM) and RNA extraction, which allows targeted mRNA analysis of immunophenotypically defined cell populations. After fixation, frozen sections are immunostained under RNAse-free conditions using a rapid three-step streptavidin-biotin technique, dehydrated and immediately subjected to LCM. RNA is extracted from captured tissue, DNAse I treated, and reverse transcribed. Acetone-, methanol-, or ethanol/acetone-fixed sections give excellent immunostaining after 12 to 25 minutes total processing time. Specificity, precision, and speed of microdissection is markedly increased due to improved identification of desired (or undesired) cell types. The mRNA recovered from immunostained tissue is of high quality. Single-step PCR is able to amplify fragments of more than 600 bp from both housekeeping genes such as beta-actin as well as cell-specific messages such as CD4 or CD19, using cDNA derived from less than 500 immunostained, microdissected cells. Immuno-LCM allows specific mRNA analysis of cell populations isolated according to their immunophenotype or expression of function-related antigens and significantly expands our ability to investigate gene expression in heterogeneous tissues.