Semi-quantitative non-radioactive in situ hybridization and its clinical application

In situ hybridization (ISH) is widely used to determine the expression of s pecific mRNA in tissues. However, the results of ISH are influenced by the amount of retained RNA in the tissue. Estimation of retained RNA in the tis sue is very important and the results of ISH should be interpreted taking i nto consideration the amount of retained RNA in samples. In the present stu dy, we determined the amount of retained RNA by performing ISH for 28S rRNA . We also analyzed the results of ISH semi-quantitatively by computer image analysis. The method was applied by calculating the ratio of specific mRNA to 28S rRNA in order to standardize the results of ISH among different tis sue samples. Our method is useful for comparing the expression of specific mRNA in different tissue samples with variable levels of retained RNA. Sinc e the conditions of fixation and storage of tissues differ among samples, e specially in clinical samples, our semi-quantitative method allows a more p recise evaluation of gene expression in different samples.