Distribution of cytokines in tumor tissues by in situ hybridization

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We established ISH technique which detect low expression of mRNAs such as cytokines using digoxigenin labeled cRNA probe in 4% paraformaldehyde-fixed and paraffin-embedded tissues. However, the sensitivity of our ISH was not enough to detect the tumor cell producing cytokine mRNA.The in situ reverse transcription-polymerase chain reaction (in situ RT-PCR) method has been used to detect low levels of expression of mRNA in cells an tissues. We applied a newly developed in situ RT-PCR to cells and quantitatively compared the sensitivity of the in situ hybridization (ISH). Human tumor cell lines with either high, moderate, low or no expression of IL-6 mRNA as determined by in vitro competitive RT-PCR were used. In situ RT-PCR was performed using Thermus thermophilus DNA polymerase special primer sets to form high molecular weight concatemers and an in situ digoxigenin (DIG) direct labeling technique. Conventional ISH was performed using DIG-labeled cRNA probe. Specific signals of IL-6 mRNA were observed in the cytoplasm of positive cells by both in situ RT-PCR and ISH. In situ RT-PCR was more than 100-fold more sensitive than ISH. The specific of in situ RT-PCR was confirmed by appropriate control tests. The data indicated that the sensitivity of our in situ RT-PCR applied to culture cells was significantly higher than that of ISH, and that the technique retained high specificity.