Qualitative and quantitative assessment of modified in situ reverse transcription-polymerase chain reaction (in situ RT-PCR) method

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 and tissues. We applied a newly developed in situ RT-PCR to cells and quant itatively compared the sensitivity of the in situ RT-PCR with that of conve ntional in situ hybridization (ISH). Human tumor cell lines with either hig h, moderate, low or no expression of IL-6 mRNA as determined by in vitro co mpetitive RT-PCR were used. In situ RT-PCR was performed using Thermos ther mophilus DNA polymerase, special primer sets to form high molecular weight concatemers and an in situ digoxigenin (DIG) direct labeling technique. Con ventional 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 si tu RT-PCR and ISH. in situ RT-PCR was more than 100-fold more sensitive tha n ISH. The specificity of in situ RT-PCR was confirmed by appropriate contr ol tests. The data indicated that the sensitivity of our in situ RT-PCR app lied to culture cells was significantly higher than that of ISH, and that t he technique retained high specificity.