Quantification of multiple mRNAs expressions in microdissected specimens : Development of liquid

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The purpose of present study is establishment of new technology, enabling quantify the expressed mRNAs in microdissected specimens. With this technique, gene expression profile in different components of the cancer may reveal. Our originally proposed method "liquid phase Northern analysis" is initiated by cDNA construction on magnetic dT beads. CDNA, fixed on beads, were then hybridized to multiple probes, each has different but distinct length. Specifically hybridized probes were then recovered, and electrophoresed on polyacrylamide gel. Each sample should present multiple ladder bands, and amount of gene expression should represented by the intensity of each band. This original method however, could not put into practical use, because of strong background noise, different hybridization capacity between different probe and/or different efficiency of reverse transcription on magnet beads. At the second year, we changed our strategy and employed multiple RNase protection assays (MRPA). When compare with conventional Northern blot analysis, MRPA is reliable enough and reproducible. Moreover, 1 MRPA assay takes the place of 7 Northen analysis. We analyzed mRNA expression of 7 different molecules, which has matrix-degradation capacity, in 10 colon cancer cell lines ; each possessed different hepatic metastasis potentials. Those cell lines with high hepatic capacity, showed negative or low expressed of MT1 -MMP and uPA.When we tried to apply this method to analyze small samples such as microdissected specimens, MRPA were again not useful.