Quantitation of NMDA receptor NR2 mRNA transcripts in human brain by competitive RT-PCR.
The NMDA-selective ionotropic receptor constitutes one of the three principal classes of L-glutamate receptors within the mammalian brain. It plays key roles in neuronal differentiation and synapse consolidation, activity-dependent forms of synaptic plasticity, and excitatory amino acid-mediated neuronal toxicity [Lab. Invest., 68 (1993) 372-387]. NMDA receptors exist as multimeric complexes comprising proteins from two families, NR1 and NR2(A-D) [J. Biol. Chem., 271 (1996) 15669-15674]. Studies on recombinant receptors have revealed that while homomeric NR2 receptors are non-functional, co-expression of an NR1 with an NR2 subunit modulates the efficacy of the resulting channel [Nature, 357 (1992) 70-74]. The RT-PCR assay we describe here was developed to allow quantitation of all hNR2 transcripts in a single-tube PCR assay. Each hNR2 isoform is quantified on the basis of standard curves in which a known amount of synthetic ribonucleic acid competitor is co-amplified against total RNA. The protocol has been applied to the quantitation of hNR2 mRNA levels in autopsy brain. Used in conjunction with a method for the quantitation of hNR1 transcripts [Brain Res. Protoc., in press], a complete analysis of NMDA receptor mRNA expression can be obtained.
Hynd
Matthew R
MR
Department of Biochemistry, University of Queensland, Brisbane, Australia.
Scott
Heather L
HL
Dodd
Peter R
PR
D017398:Alternative Splicing / Q000235:genetics; D001921:Brain / Q000378:metabolism*; D001923:Brain Chemistry / Q000235:genetics*; D003001:Cloning, Molecular; D018698:Glutamic Acid / Q000378:metabolism; D006624:Hippocampus / Q000378:metabolism; D006801:Humans; D009044:Motor Cortex / Q000378:metabolism; D009474:Neurons / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D016194:Receptors, N-Methyl-D-Aspartate / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D013569:Synapses / Q000235:genetics / Q000378:metabolism; D009435:Synaptic Transmission / Q000235:genetics; D013698:Templates, Genetic
C120997:NR2A NMDA receptor; D020033:Protein Isoforms; D012333:RNA, Messenger; D016194:Receptors, N-Methyl-D-Aspartate; D018698:Glutamic Acid