Improvement of ISH method for the detection of rare mRNAs in pathology archival tissues and establishment of its standard evaluation method

病理档案组织中稀有mRNA检测ISH方法的改进及其标准评价方法的建立

基本信息

批准号：
15590315
负责人：
SATO Yuichi
金额：
$ 2.24万
依托单位：
Kitasato University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

项目摘要

The specific protein expression has been semi-quantitatively examined by immunostaining. No in situ hybridization (ISH) method to detect the expression of mRNAs semi-quantitatively has been reported. In this study, to examine the correlation of the fixation conditions and proteinase K treatment, house keeping gene such as beta-2-microglobulin (beta-2M) mRNA was detected by ISH method using one day to 16 days formalin-fixed and paraffin-embedded normal human tissues. As a result, the expression of beta-2M was most preserved in all but heart tissue fixed by formalin for one day. And the expression levels tend to be reduced during elongation of fixation time. Furthermore, optimal proteinase K treatments were different in each tissue that was fixed even if in the same conditions. We also demonstrate that the expression levels of tissue sections that stored at 4 C for a long time were decreased than those in tissue sections just after cutting. To raise detection sensitivity, the rapid ISH method using cRNA probes has been improved. Using this modified method, we were able detect insulin and glucagon mRNAs clearly within three hours. The rapid ISH with cRNA probes could have many applications in various research fields and clinical laboratories. The expressions of autocrine motility factor (AMF) and its receptor gp78 mRNA were detected in formalin-fixed and paraffin-embedded pathology archival high-grade astrocytoma tissues by the highly sensitive ISH method. The positive rate of AMF mRNA was significantly higher than in anaplastic astrocytomas. The overall survival of patients with AMF expression was significantly shorter than patients without AMF expression. In anaplastic astrocytomas, the overall survival of patients with AMF expression was also significantly shorter than patients without AMF expression. This study demonstrated that AMF is a poor prognostic factor in high-grade astrocytomas.

特异性蛋白表达已通过免疫染色进行半定量检查。据报道，没有原位杂交方法（ISH）方法可检测MRNA半量化的表达。在这项研究中，为了检查固定条件和蛋白酶K治疗的相关性，使用一天到16天福尔马林固定和波帕蛋白 - 嵌入正常的人体组织。结果，除了福尔马林固定的心脏组织以外，β-2M的表达最多。在固定时间伸长期间，表达水平往往会降低。此外，即使在相同条件下，最佳蛋白酶K处理也不同。我们还证明，与切割后的组织切片相比，在4 C下储存长时间的组织切片的表达水平降低了。为了提高检测灵敏度，使用CRNA探针的快速ISH方法得到了改善。使用这种修饰方法，我们能够在三个小时内清楚地检测到胰岛素和胰高血糖素mRNA。带有CRNA探针的快速ISH可以在各个研究领域和临床实验室中使用许多应用。通过高度敏感的ISH方法，在福尔马林固定和石蜡包含的病理学档案档案中检测到自分泌运动因子（AMF）及其受体GP78 mRNA的表达。 AMF mRNA的正速率显着高于变性星形胶质细胞瘤。与没有AMF表达的患者相比，AMF表达患者的总生存率明显短。在那内胶质细胞瘤中，与没有AMF表达的患者相比，AMF表达患者的总体存活率也明显短。这项研究表明，AMF在高级星形胶质细胞瘤中是一个差的预后因素。