Quantitation of nicotinic acetylcholine receptor subunits alpha 4 and beta 2 messenger RNA in postmortem human brain using a non-radioactive RT-PCR and CCD imaging system.
We present a simple and rapid procedure for quantifying mRNA in the brain after RT-PCR, in which the intensity of the ethidium bromide luminescence of PCR products is measured directly from electrophoretic gels by a highly sensitive CCD camera combined with an image analyzing computer system (Gel Doc 1000 system). The CCD camera allows the mRNA in the ethidium bromide-stained PCR-amplified bands to be quantified in a broad exponential range of PCR cycles. The proposed protocol enables standard curves to be constructed to examine the relationship between the number of reaction cycles and amplified log intensity and between the amount of sample RNA for RT-PCR and amplified intensity. The method was applied to nicotinic acetylcholine receptor (nAChR) subunits alpha 4 and beta 2 mRNA in postmortem human putamen in the present study, but is also applicable to mRNAs of other receptors and neurotransmitter precursor peptides.
Utsugisawa
K
K
Department of Neurology, Iwate Medical University, Morioka, Japan.
Tohgi
H
H
Yoshimura
M
M
Nagane
Y
Y
Ukitsu
M
M
D000199:Actins / Q000235:genetics; D001921:Brain / Q000378:metabolism*; D002102:Cadaver; D006801:Humans; D007091:Image Processing, Computer-Assisted; D010781:Photography / Q000295:instrumentation; D012333:RNA, Messenger / Q000378:metabolism*; D011978:Receptors, Nicotinic / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D000199:Actins; D012333:RNA, Messenger; D011978:Receptors, Nicotinic