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

本研究的目的是建立一种新的技术，使微解剖标本中表达的mrna能够量化。通过这项技术，基因表达谱在癌症的不同组成部分可能揭示。我们最初提出的“液相北方分析”方法是通过在磁性dT珠上构建cDNA而发起的。CDNA固定在小珠上，然后杂交到多个探针上，每个探针都有不同但不同的长度。然后回收特异性杂交探针，并在聚丙烯酰胺凝胶上电泳。每个样本应呈现多个阶梯带，每个阶梯带的强度代表基因表达量。但由于背景噪声强、不同探针间杂交能力不同以及磁珠上逆转录效率不同等原因，该方法无法投入实际应用。第二年，我们改变了策略，采用了多种rna酶保护试验（MRPA）。与传统的Northern blot分析相比，MRPA具有足够的可靠性和可重复性。此外，1个MRPA分析取代了7个Northen分析。我们分析了10种结肠癌细胞系中7种具有基质降解能力的不同分子的mRNA表达；每一种都具有不同的肝转移潜能。高肝容量的细胞系MT1 -MMP和uPA均呈阴性或低表达。当我们试图将这种方法应用于分析小样本，如显微解剖标本时，MRPA再次不起作用。

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.