中华皮肤科杂志 ›› 2000, Vol. 33 ›› Issue (2): 80-82.

• 论著 • 上一篇    下一篇

cDNA代表性差异分析法的建立及克隆银屑病相关基因片段初探

陶苏江1, 范青源1, 方跃明1, 谢毅2, 应康2, 牟贤龙1, 顾军1, 郑茂荣1   

  1. 1. 第二军医大学附属长海医院皮肤科, 上海 200433;
    2. 复旦大学遗传工程国家重点实验室
  • 收稿日期:1999-03-05 发布日期:2000-04-15

Cloning of Psoriasis-associated Genes by Representational Difference Analysis of cDNA

TAO Sujiang1, FAN Qinyuan1, FANG Yueming1   

  1. Department of Dermatology, Changhai Hospital, Second Military Medical University, Shanghai 200433
  • Received:1999-03-05 Published:2000-04-15

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摘要： 目的 通过cDNA代表性差异分析法(cDNA-representational difference analysis,RDA)来寻找银屑病皮损组织中特异表达的基因片段。方法 用改良的异硫氰酸胍一步法提取银屑病皮损及正常皮肤组织的tRNA及mRNA,逆转录后采用差减杂交及选择性PCR扩增相结合为基础的RDA方法,使含有特定接头的银屑病组cDNA片段得到大量扩增。结果 通过质量分数1%琼脂糖凝胶电泳及放射自显影分析,两组皮肤组织mRNA质量及丰度都比较高;经过两次PCR扩增后在琼脂糖凝胶电泳上可见5条清晰条带,其大小范围在100～600bp之间。结论 本实验表明银屑病皮损组织中可能存在与正常皮肤组织差异表达的基因;cDNA-RDA方法对于识别差异表达的基因具有高效性。

关键词: 银屑病, 聚合酶链反应, 核酸杂交

Abstract: Objective To identify the specifically expressed genes in psoriatic lesions by cDNA-representational difference analysis(cDNA-RDA).Methods The total RNA from psoriatic lesions and normal skin was extracted with modified single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. mRNA was converted to cDNA. Then cDNA-RDA was employed with a PCR-coupled subtractive approach. Only psoriatic cDNA which contained appropriate adaptors could be amplified. Results The high quality and abundance of mRNA from both lesions and normal skin were demonstrated by 1% agarose gel electrophoresis and autoradiography.After double amplification,the PCR products showed 5 distinctive bands on 1% agarose gel ranged from 100 to 600 bp. Conclusions Our experiment demonstrates that differentially expressed genes might present in psoriatic lesions in comparison with that in normal skin. cDNA-RDA is a highly efficient method for identification of such differentially expressed genes.

Key words: Psoriasis, Polymerase chain reaction, Subtractive hybridization