Szakacs J G, Livingston S K
Department of Pathology, University of South Florida, College of Medicine, Tampa 33612.
Ann Clin Lab Sci. 1994 Jul-Aug;24(4):324-38.
It is now possible to detect low copy numbers of messenger ribonucleic acid (mRNA) while retaining good histologic morphology for the determination of specific gene expression in diseased tissues. This technology will allow the pathologist to provide important prognostic information about tumors (expression of oncogenes and growth factors), to identify the subclones within the tumor which may be most likely to metastasize (expression of adhesion molecules and proteases) and to identify etiologic genetic aberrations (viral insertions). A technique for in-situ hybridization to mRNA has been developed for use in formalin fixed paraffin embedded tissues which is suitable for a hospital histology laboratory. Optimal conditions for the procedure were determined by using a biotinylated poly (d)T oligonucleotide probe. Results were dependent on the tissue type, fixation time, condition of the tissue prior to fixation, and degree of digestion before hybridization. The temperature and conditions of hybridization were optimized so that the poly d(T) control probe and the longer test probe could be run simultaneously. Streptavidin and avidin alkaline phosphatase detection systems were tested using levamisole to minimize background staining, and a biotin blocking agent to reduce reaction to renal tubular biotin. Increasing the temperature of stringency washes did not significantly improve the specificity but had a markedly detrimental effect on tissue morphology. The mRNA appears to remain stable within routinely fixed surgical material over long periods of time allowing for large retrospective studies. A review of c-erbB-2 expression in 16 human breast lesions was carried out comparing mRNA in-situ hybridization to immunoperoxidase and cytosolic methods. By direct localization of both message and antigen, it was possible to demonstrate focal positivity that cytosolic methods did not detect. Aberrant translation was noted in one case, and c-erbB-2 expression in non-malignant breast was detected in two cases.
现在可以检测信使核糖核酸（mRNA）的低拷贝数，同时保留良好的组织形态学，以确定病变组织中的特定基因表达。这项技术将使病理学家能够提供有关肿瘤的重要预后信息（癌基因和生长因子的表达），识别肿瘤内最有可能转移的亚克隆（黏附分子和蛋白酶的表达），并识别病因性基因畸变（病毒插入）。已开发出一种用于福尔马林固定石蜡包埋组织的mRNA原位杂交技术，适用于医院组织学实验室。通过使用生物素化的聚（d）T寡核苷酸探针确定了该程序的最佳条件。结果取决于组织类型、固定时间、固定前组织的状况以及杂交前的消化程度。对杂交温度和条件进行了优化，以便聚d（T）对照探针和较长的测试探针可以同时运行。使用左旋咪唑测试链霉亲和素和抗生物素蛋白碱性磷酸酶检测系统以尽量减少背景染色，并使用生物素阻断剂减少对肾小管生物素的反应。提高严格洗涤的温度并没有显著提高特异性，但对组织形态有明显的不利影响。mRNA似乎在常规固定的手术材料中长时间保持稳定，这使得进行大规模回顾性研究成为可能。对16例人类乳腺病变中的c-erbB-2表达进行了综述，比较了mRNA原位杂交与免疫过氧化物酶和胞质方法。通过对信息和抗原的直接定位，有可能证明胞质方法未检测到的局灶性阳性。在1例中发现异常翻译，在2例非恶性乳腺中检测到c-erbB-2表达。
