一种新型的基因表达水平检测方法

Validation of a New Semi-quantitative RT-PCR Via Competitively Binding to Avidin by Biotin Labeled RT-PCR Product and Horseradish Peroxidase

目的 :建立一种简便、特异的检测组织血管紧张素Ⅱ (angiotensinⅡ ,AngⅡ )受体基因表达变化的半定量逆转录 聚合酶链反应 (reversetranscription polymerasechainreaction ,RT PCR)技术。方法 :采用异硫氰酸胍 酚 氯仿法提取组织总RNA ,用生物素 (biotin ,B)标记的AngⅡ受体的特异引物进行RT PCR扩增 ,PCR产物纯化后 ,再与过氧化物酶标记生物素竞争性地结合包被到酶标板上的亲和素 (avidin) ,经过显色反应并测定其吸光度值可反映特异性PCR产物量 ,从而推断模板核酸中AngⅡ受体基因的相对含量。结果 :异硫氰酸胍 酚 氯仿法提取组织总RNA的质量较高 ;PCR的最适反应条件为 94℃变性 30s、5 9℃退火 40s、72℃延伸 6 0s,循环 33次 ,最后 72℃延伸 5min ;酶标仪所测阴性对照和扩增产物光密度之差能够反映不同模板中AngⅡ受体基因的相对含量。结论 :该方法是一种简便、特异、可行的半定量RT

Abstract: To establish a simple, special and semi quantitative reverse transcription polymerase chain reaction technique for detecting the gene expression of AngⅡreceptors. Methods: Total RNAs were extracted quickly by guanidnium phenol chloroform method from tissues. The premier was modified so that amplified products were labeled with biotin. When purified amplified products and the horseradish peroxidase labeled with biotin were diluted and synchronously added into the steptavidin coated wells, biotinylated complexes bound to the avidin competitively. After color reaction, the data was detected by a microplate reader and reflected the quantity of amplified products, from which we could deduce the relative level of the gene expression of angiotensin Ⅱreceptors. Results: The guanidnium phenol chloroform method was suitable for extraction total RNAs from tissues. Optimal reaction conditions for PCR were 33 cycles of 94 ℃ for 30 seconds, 59 ℃ for 40 seconds, and 72 ℃ for 60 seconds. The difference between the negative control and amplified products in optical density(OD) detected by the microplate reader could reflect the relative level of the gene expression of angiotensin Ⅱreceptors. Conclusion: This established assay is a simple, sensitive, and specific quantitative reverse transcription polymerase chain reaction for detecting gene expression.