[Quantitative analysis of mRNA level by PCR method using single basemutated template as inner standard].
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 microgram of total RNA isolated from liver tissue of Sprague Dawley rats was about 1.25 x 10(-20) mol.
Lu
L M
LM
Department of Physiology, State Key Laboratory of Medical Neurobiology, Shanghai Medical University.
Li
H Y
HY
Wang
R
R
Yao
T
T
D000818:Animals; D018076:DNA, Complementary / Q000235:genetics; D008099:Liver / Q000378:metabolism; D008297:Male; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D017483:Receptors, Vasopressin / Q000235:genetics*
D018076:DNA, Complementary; D012333:RNA, Messenger; D017483:Receptors, Vasopressin