Measurement of dorsal root ganglion P2X mRNA by SYBR Green fluorescence.
The P2X receptor is a receptor-gated cationic channel that responds to ATP. The quantification of P2X mRNA expression in dorsal root ganglion (DRG) provides important information for neuropathic pain studies. We developed a rapid and sensitive external-standard-based real-time quantitative PCR assay for the quantification of mRNA of P2X receptors in mouse tissue samples. The assay uses a double-stranded DNA fluorescent dye, SYBR Green I, to continuously monitor product formation with a GeneAmp 5700 Sequence Detection System (PE Applied Biosystems). To establish the quantitative PCR amplification in a wide range of target transcripts, optimum parameters of primer sequences, concentrations of primers and/or templates, and PCR thermal protocols were experimentally determined. We also tested the reliability of this method in established experimental murine models, which were made by ligation or cutting down of the sciatic nerve. The parameters defined in this assay should be applicable to the quantification of other types of pain models and other tissue samples of mouse.
Ueno
Shinya
S
Department of Pharmacology, School of Medicine, Fukuoka University, 7-45-1 Nanakuma Jonan-ku, Fukuoka 814-0180, Japan. shinyau@hama-med.ac.jp
Yamada
Hiromi
H
Moriyama
Tomoko
T
Honda
Kenji
K
Takano
Yukio
Y
Kamiya
Hiro-o
HO
Katsuragi
Takeshi
T
D000818:Animals; D052160:Benzothiazoles; D003959:Diamines; D004195:Disease Models, Animal; D005456:Fluorescent Dyes; D005727:Ganglia, Spinal / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D009409:Nerve Crush; D009437:Neuralgia / Q000235:genetics / Q000378:metabolism / Q000503:physiopathology; D009475:Neurons, Afferent / Q000166:cytology / Q000378:metabolism*; D009930:Organic Chemicals; D010523:Peripheral Nervous System Diseases / Q000235:genetics / Q000378:metabolism / Q000503:physiopathology; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D011804:Quinolines; D012333:RNA, Messenger / Q000032:analysis*; D018048:Receptors, Purinergic P2 / Q000235:genetics*; D058469:Receptors, Purinergic P2X; D012584:Sciatic Nerve / Q000293:injuries / Q000601:surgery
D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; D012333:RNA, Messenger; D018048:Receptors, Purinergic P2; D058469:Receptors, Purinergic P2X; C098022:SYBR Green I