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

通过免疫染色对特异性蛋白质表达进行了半定量检查。目前尚未报道半定量检测mRNA表达的原位杂交（ISH）方法。在本研究中，为了检查固定条件和蛋白酶K处理的相关性，使用1天至16天福尔马林固定和石蜡包埋的正常人体组织，通过ISH方法检测管家基因，例如β-2-微球蛋白（β-2M）mRNA。结果，除了用福尔马林固定一天的心脏组织外，β-2M 的表达在除心脏组织外的所有组织中都得到了最完整的保存。并且随着固定时间的延长，表达水平趋于降低。此外，即使在相同条件下，每个固定组织的最佳蛋白酶 K 处理也不同。我们还证明，长期保存在4℃的组织切片的表达水平比刚切割后的组织切片的表达水平有所降低。为了提高检测灵敏度，对使用 cRNA 探针的快速 ISH 方法进行了改进。使用这种改进的方法，我们能够在三小时内清楚地检测到胰岛素和胰高血糖素 mRNA。使用 cRNA 探针的快速 ISH 可以在各个研究领域和临床实验室中有许多应用。采用高灵敏ISH方法检测福尔马林固定石蜡包埋的病理档案高级别星形细胞瘤组织中自分泌运动因子（AMF）及其受体gp78 mRNA的表达。 AMF mRNA阳性率明显高于间变性星形细胞瘤。有AMF表达的患者总生存期明显短于无AMF表达的患者。在间变性星形细胞瘤中，有 AMF 表达的患者的总生存期也显着短于没有 AMF 表达的患者。这项研究表明，AMF 是高级别星形细胞瘤的不良预后因素。

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.