mRNA差异显示方法的建立及条件优化  

PAGESS Establishment and optimization of mRNA differential-PCR

作　　者：王小伟[1] 孙俊红[1] 籍晓元[1] 杜秋香[1] 王英元[1] 

机构地区：[1]山西医科大学法医学院病理学教研室,太原030001

出　　处：《山西医科大学学报》2008年第4期289-292,共4页Journal of Shanxi Medical University

基　　金：山西省留学回国人员科研基金资助项目(200528);山西省青年科研基金资助项目(2007021047)

摘　　要：目的研究大鼠皮肤肌肉特异性基因的表达,探讨引物的不同、退火温度变化与扩增产物特异性之间的关系。方法12只健康雄性Sprague-Dawley大鼠分为正常对照组与实验组(挫伤4h)。分别提取总RNA,反转录成cDNA,通过不同引物、退火温度及反应体系进行PCR产物扩增,比较不同条件下差异条带的情况。结果总RNA量在2-4μg时,扩增条带最亮;提高反转录温度有利于长片段cDNA的合成;使用TaKaRa公司带有T7启动子的OligodT12NM和M13的随机引物时,可最大限度地减少非特异性条带;先使用低严谨的退火温度,再使用高严谨的退火温度可显著地增强差异条带重复性和敏感性。结论差异显示技术方法简便、灵敏、高效,适用于一般性的基因差异表达研究。Objective To study skin- and muscle-specific gene expression, and to explore the relationship between different primers, annealing temperatures and specific amplification in rats. Methods Twelve healthy male Sprague-Dawley rats were randomly divided into two groups： control group（ n = 6） and contusion group（ n = 6）. Total RNA was extracted, and PCR product was reversely transeripted into cDNA using different primers at different armealing temperatures in different reaction system. Results Amplification was the highest when total RNA was 2 - 4 μg. The increase of reverse transcription temperature was useful for large eDNA fragment synthesis. Oligonucleotide dT12 NM with T7 promoter and random primers with M13 maximized the reduction of non-specific bands. Annealing temperatures significantly enhanced sensitivity and reproducibility of differences. Conclusion Differential display is simple, sensitive, efficient to gene differential expression.

关 键 词：差异显示技术 锚定引物 随机引物 退火温度 

分 类 号：Q789[生物学—分子生物学]