• 我要登录|
  • 免费注册
    |
  • 我的丁香通
    • 企业机构:
    • 成为企业机构
    • 个人用户:
    • 个人中心
  • 移动端
    移动端
丁香通 logo丁香实验_LOGO
搜实验

    大家都在搜

      大家都在搜

        0 人通过求购买到了急需的产品
        免费发布求购
        发布求购
        点赞
        收藏
        wx-share
        分享

        Gene Expression Analysis by CD-RT-PCR Eric de Kant

        互联网

        560
        With the introduction of polymerase chain reaction (PCR), analysis on even minute amounts of DNA and RNA from biological samples became practicable. Conventional methods of mRNA analysis are often not sensitive enough for detection of low-abundance transcripts or broad examination of gene expression in small amounts of RNA. Quantitative mRNA characterization by reverse transcription (RT) of RNA and subsequent PCR (RT-PCR) is, however, compared to qualitative RT-PCR detection of RNA, more complicated because of two features inherent in in vitro ampllfication. First, during the exponential phase, minute differences in a number of variables can greatly influence reaction rates, with substantial effect on the yield of PCR products, Second, as a consequence of the consumption of reaction components and generation of inhibitors, the amplification enters a plateau phase. At this point, the reaction rate declines to an unknown level. Another source of errors in quantitative RT-PCR analysis lies in the determination of the amount of RNA to be analyzed for each sample. In small samples, the total amount of RNA may even be beyond the limit of detection. The sample loading problem can be solved by presenting the level of expression of the gene of interest in reference to a constitutively expressed gene. In PCR, this can be done by the simultaneous amplification of two different genes in one reaction vessel, which is called differential PCR (1 ). However, in many cases a quantitative PCR assay is desired that is internally controlled both for errors in comparison between samples and for the efficiency of the amplification reaction. To this end, a technique was devised that combines competitive PCR and differential RT-PCR by co-amplification of two genes and their corresponding competitive templates (2 ). This chapter describes the working procedures for that complete assay, called competitive and differential RT-PCR (CD-RT-PCR) and concomitant techniques.
        ad image
        提问
        扫一扫
        丁香实验小程序二维码
        实验小助手
        丁香实验公众号二维码
        扫码领资料
        反馈
        TOP
        打开小程序