Bresser J, Evinger-Hodges M J
Department of Clinical Immunology, University of Texas M. D. Anderson Hospital and Tumor Institute, Houston 77030.
Gene Anal Tech. 1987 Sep-Oct;4(5):89-104. doi: 10.1016/0735-0651(87)90002-1.
This paper describes methods that are commonly used for performing mRNA in situ hybridizations. Each stage of the procedure has been analyzed to identify the parameters that most significantly affect the final cell morphology and sensitivity of the system. We have identified key elements of the procedure as the fixation employed, the type of polynucleotide probe and label chosen, and the detection system used. By optimizing these critical components, we have developed a procedure for performing mRNA in situ hybridizations that takes 2-4 hours and has a sensitivity of 1-10 molecules of mRNA per cell. This system has been used to detect levels of oncogene expression in normal bone marrow and peripheral blood. It is possible to detect the expression of three oncogenes (c-myc, c-sis, and c-abl) simultaneously in a small population of cells from the peripheral blood of leukemic patients.
本文描述了常用于进行mRNA原位杂交的方法。对该程序的每个阶段进行了分析，以确定对最终细胞形态和系统灵敏度影响最大的参数。我们已经确定该程序的关键要素为所采用的固定方法、选择的多核苷酸探针和标记类型以及所使用的检测系统。通过优化这些关键成分，我们开发了一种进行mRNA原位杂交的程序，该程序耗时2 - 4小时，灵敏度为每个细胞1 - 10个mRNA分子。该系统已用于检测正常骨髓和外周血中癌基因的表达水平。有可能在白血病患者外周血的一小群细胞中同时检测三种癌基因（c-myc、c-sis和c-abl）的表达。
