Localisation of GABA(A) receptor subunits in the CNS using RT-PCR.
This paper presents an RT-PCR in situ histochemistry (RT-ISH) method for the detection and localisation of isoforms of the GABA(A) receptor in formalin-fixed, paraffin-embedded brain sections. RT-ISH was performed using PCR conditions already established in our laboratory for the amplification of the alpha(1-3) and beta(1-3) subunits of the GABA(A) receptor [2,5]. Initial experiments determined whether mRNA isolated from such sections was suitable for use in RT-PCR. Transcripts encoding both the alpha- and beta-subunits of the GABA(A) receptor were successfully amplified. RT-ISH, a one-step RT-PCR method, was used to amplify the transcripts and digoxigenin-labeled nucleotides were directly incorporated into the amplified products during cycling. RT-PCR products were detected using anti-digoxigenin antibody conjugated to alkaline-phosphatase and signal was visualised using light microscopy. This protocol may be used to study the expression of GABA(A) receptor isoforms in vivo and examine alterations in receptor composition during development and disease states.
Hawkins
A J
AJ
Neuroscience Unit, Department of Biochemistry, University of Queensland, St, 4072, Lucia, Australia. hawkins@biosci.uq.edu.au
Dodd
P R
PR
D001933:Brain Stem / Q000378:metabolism*; D005404:Fixatives; D005557:Formaldehyde; D006801:Humans; D017403:In Situ Hybridization; D016612:Paraffin Embedding; D020033:Protein Isoforms / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011963:Receptors, GABA-A / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014018:Tissue Distribution
D005404:Fixatives; D020033:Protein Isoforms; D012333:RNA, Messenger; D011963:Receptors, GABA-A; D005557:Formaldehyde