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In situ hybridization (ISH) is widely used to determine the expression of specific 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 tissue is very important and the results of ISH should be interpreted taking into consideration the amount of retained RNA in samples. In the present study, 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 tissue samples. Our method is useful for comparing the expression of specific mRNA in different tissue samples with variable levels of retained RNA. Since the conditions of fixation and storage of tissues differ among samples, especially in clinical samples, our semi−quantitative method allows a more precise evaluation of gene expression in different samples

Semi-Quantitative Non-Radioactive In Situ Hybridization and Its Clinical Application.