Novel quantitative reverse-transcribed polymerase chain reaction of mu opioid receptor mRNA level.
Reverse-transcribed polymerase chain reaction (RT-PCR) can quantify gene transcripts at low levels and in small samples. Semi-quantitative and quantitative RT-PCR has significant advantages over traditional RNA assays, such as Northern blotting and ribonuclease protection assay. However, owing to the exponential nature of PCR, considerable effort is required to verify linearity of the reaction. Thus, care must be taken to detect small but physiologically relevant changes in gene expression. Using a rapid and highly sensitive RT-PCR method, TaqMan real-time RT-PCR, we determined agonist-induced changes in rat mu opioid receptor (MOR) mRNA levels in cultured cells and compared our results with those obtained by radiolabeled quantitative RT-PCR, which is also highly sensitive but much more time-consuming than TaqMan RT-PCR. Both methods showed up-regulation of agonist-induced MOR. TaqMan RT-PCR showed a similar sensitivity to radiolabeled quantitative RT-PCR and is suitable for the measurement of large numbers of samples. Moreover, no need for radiolabeled compounds is also an advantage of TaqMan PCR. This protocol will probably be useful for quantifying MOR in animal and human tissues.
Yoshikawa
M
M
Department of Anesthesiology, Nara Medical University, 840 Shijo-cho, Kashihara, 634-8522, Nara, Japan. ymayumi@nmu-gw.cc.naramed-u.ac.jp
Nakayama
H
H
Ueno
S
S
Nishimine
N
N
Furuya
H
H
D000701:Analgesics, Opioid; D000818:Animals; D000242:Cyclic AMP; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D005283:Fentanyl; D015870:Gene Expression; D016716:PC12 Cells; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017450:Receptors, Opioid, mu / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019914:Taq Polymerase
D000701:Analgesics, Opioid; D012333:RNA, Messenger; D017450:Receptors, Opioid, mu; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D000242:Cyclic AMP; D019914:Taq Polymerase; D005283:Fentanyl