摘要
ｍＲＮＡ差异显示技术是分离差异表达基因的强有力工具。本工作旨在优化无同位素的银染差异显示方法。以总ＲＮＡ为模板，用锚定引物与随机引物的组合进行ＲＴ－ＰＣＲ反应，ＰＣＲ扩增产物在变性的聚丙烯酰胺凝胶上电泳。用银染方法显示电泳ｃＤＡＮ条带，其结果是：（１）ＲＮＡ加入量为１～３μｇ，ＲＴ－ＰＣＲ反应能扩增出较多的ｃＤＮＡ条带，而低于０．５μｇ时扩增条带数目较少；（２）在３６～４２℃的范围内，随着温度的增加，扩增的条带减少；而４２℃时扩增的条带数目锐减，特别是当ＲＮＡ加入量小于１μｇ时几乎无条带显示；３６℃扩增的条带过多而趋于ｓｍｅａｒ；（３）优化了银染液程序，染色液和显色液中３７％甲醛的量分别为０．０３％～０．０５％和０．０７５％～０．１％，显色温度４～１２℃时，显示出清晰的条带；（４）用此方法获得数条电针镇痛有效与无效大鼠表达差异的ｃＤＮＡ条带。总之，通过改变参数条件，优化了快速简便的银染ｍＲＮＡ差异显示方法。
mRNA differential display is a powerful instrument to identify differentially expressed genes.This work is designed to optimize a non radioactive differential display with silver staining.Total RNA as template was reverse transcribed into corresponding cDNAs by 3′ anchor primer and the cDNAs were amplified by polymerase chain reaction(PCR) with a set of one same 3′ anchor primer and one 5′ arbitrarily primer.The PCR products were then resolved on denaturing polyacylamimide gel. cDNA bands were visualized by silver staining.The results revealed as follows: (1)The amount of RNA used was 1～3 μg,which produced many cDNA bands by RT PCR. Few of bands were observed when the amount of RNA was reduced to less than 0.5 μg.(2)cDNA bands were decreased with increasing temperature in a range of 36～42℃.(3) Silver staining procedure was modified.The concentration of 37% formaldehyde in staining solution and developing solution was optimized,and the developing temperature was appropriate at 4～12℃.(4) Several cDNA fragments were identified from brain of responder and non responder rat for electroacupuncture induced analgesia.In conclusion,the silver staining mRNA differential display optimized in parameters is a rapid and easy procedure.
基金
国家自然科学基金
关键词
MRNA
差异显示
聚合酶链反应
银染
基因表达
mRNA differential display reverse transcription polymerase chain reaction silver staining gene expression
