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

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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.