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采用单碱基突变模板作为内对照对组织中mRNA水平进行PCR定量

陆利民, 李海雁, 汪蓉, 姚泰

上海医科大学生理学教研室,医学神经生物学国家重点实验室.上海 200032

摘要

用PCR方法对原始模板进行单碱基突变,在原始模板DNA的特定位点引入一个EcoR I酶切位点。单碱基突变的DNA经PCR扩增后定量、稀释,作为内标加入到样品中与待测DNA同时进行PCR扩增,扩增产物经酶切后电泳,根据电泳结果中不同分子量DNA片段的含量,对样品中待测基因拷贝数进行定量分析。实验结果观察到：每1#mu#g肝脏组织总RNA经逆转录后AVPV1受体cDNA拷贝数约1.25×10~(-20)mol。

关键词： 定量; PCR; 基因突变; 血管升压素; V1受体

Quantitative analysis of mRNA level by PCR method using single basemutated template as inner standard

Lu Limin, Li Haiyan, Wang Rong, Yao Tai

Department of Physiology, State Key Laboratory of Medical Neurobiology, Shanghai Medical University. Shanghai 200032

Abstract

Using simple polymerase chain reaction (PCR) method, a single base changed mutant was generated, to which an EcoR I restriction site was added at a specific position of the primer template. The copies of the mutant could be quantified after amplification by PCR. Then the diluted mutant used as an inner standard was added into the samples to be detected. In the same PCR reaction, the mutated and primer template DNA were amplified. After digestion by EcoR I, the PCR products were electrophoresed in 2% agarose gel. The DNAs of different size were separated by electrophoresis and the copies of the primer template in the samples was quantified. The experimental results showed that the cDNA copies of AVP V1 receptor in reverse transcription products from 1 #mu#g of total RNA isolated from liver tissue of Sprague Dawley rats was about 1.25×10~(-20)mol.

Key words: Quantitative;PCR;Genetic mutant;Vasopressin;V1 receptor

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引用本文：

陆利民, 李海雁, 汪蓉, 姚泰. 采用单碱基突变模板作为内对照对组织中mRNA水平进行PCR定量[J]. 生理学报 1997; 49 (2): .

Lu Limin, Li Haiyan, Wang Rong, Yao Tai. Quantitative analysis of mRNA level by PCR method using single basemutated template as inner standard. Acta Physiol Sin 1997; 49 (2): (in Chinese with English abstract).