权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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

显微解剖标本中多种 mRNA 表达的定量：液体的开发

基本信息

批准号：
10557119
负责人：
NOGUCHI Masayuki
金额：
$ 2.37万
依托单位：
Univ. of Tsukuba
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10557119/
关键词：
mRNA RNAse protection assay Colon cancer MMP 定量

项目摘要

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.

本研究的目的是建立一种新的技术，使定量表达的mRNA在显微解剖标本。通过这项技术，可以揭示癌症不同组成部分的基因表达谱。我们最初提出的方法“液相北方分析”是通过在磁性dT珠上构建cDNA来启动的。然后将固定在珠上的cDNA与多个探针杂交，每个探针具有不同但不同的长度。然后回收特异性杂交的探针，并在聚丙烯酰胺凝胶上电泳。每个样品应呈现多个梯状条带，每个条带的强度应代表基因表达量。然而，由于强背景噪声、不同探针之间的不同杂交能力和/或磁珠上的不同逆转录效率，这种原始方法不能投入实际使用。在第二年，我们改变了策略，采用了多种RNase保护试验（MRPA）。与传统的北方印迹法相比，MRPA具有较高的可靠性和重复性。此外，1个MRPA测定取代了7个Northen分析。我们分析了7种不同分子的mRNA表达，这些分子具有基质降解能力，在10个结肠癌细胞系中，每个细胞系具有不同的肝转移潜能。当我们试图将这种方法应用于小样本分析时，如显微解剖标本，MRPA同样不能起作用。