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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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Citing This Article：

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).