Simple quantitative reverse transcribed-polymerase chain reaction (RT-PCR) method involving recombinant RNA generated by a false-priming PCR product.
A simple and efficient method for preparing rcRNA through competitive RT-PCR has been developed. The basis of this method is the use of a false-priming PCR product including the same primers as a specific product. A 290 bp fragment obtained by two-step PCR was subcloned into a plasmid vector and then the cloned DNA was transcribed into rcRNA. After competitive RT-PCR using sample RNA and rcRNA had been carried out, Southern blot hybridization was performed. The method was applied to determine the amounts of PIMT mRNAs in the rat pituitary. The quantitative analysis indicated that an at least 2-fold difference in PIMT mRNA level can be accurately determined with our method.
Sato
M
M
Department of Medicine, Kagawa Medical School.
Mizobuchi
M
M
Murao
K
K
Tamaki
M
M
Takahara
J
J
D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D017931:DNA Primers; D008969:Molecular Sequence Data; D010902:Pituitary Gland / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods*; D012313:RNA / Q000096:biosynthesis / Q000235:genetics*; D051381:Rats; D014158:Transcription, Genetic
D017931:DNA Primers; D012313:RNA; D004247:DNA