２次元ディファレンシャルディスプレイ法を用いた甲状腺癌における遺伝子発現の解析

Abstract

We have developed a novel two-dimensional display method of cDNA and compared the gene expression profile between human normal thyroid and papillary carcinoma. The cDNAs were synthesized with a Not I anchor primer from mRNAs prepared from surgery materials. The cDNAs were digested with three restriction enzymes, Not I, Eco RV, and Pvu II. The protruding Not I ends were filled in with 32P deoxynucleotides and the isotope-labeled cDNA fragments were separated into two dimensions. The cDNAs were in situ digested with Hinf I before the second dimension electrophoresis. Using our conditions, approximately 500 cDNA fragments were visualized as major discrete spots on a single autoradiogram without probes. We cloned two cDNA spots selected from among a number of spots which showed apparently different intensities between normal thyroid and papillary carcinoma. One spot (Nc-4) was more intense in the normal tissue than in the tumor and the other (Pc-1) was more intense in the tumor. A Northern blot analysis with clone Nc-4 showed high expression in a normal thyroid sample but low expression in a papillary carcinoma sample. A Northern blot analysis with the Pc-1 clone confirmed the over-expression of this mRNA in papillary carcinomas. This method of separating the labeled cDNA into two dimensions is much more accurate and powerful than conventional differential display methods. In addition, this method is quite rapid; one can conduct a series of experiments, gel preparation and cloning, within about 10 days. We believe that this approach, in combination with 2D genomic DNA analysis, is useful for cancer research.