Quantitation of alternatively spliced NMDA receptor NR1 isoform mRNA transcripts in human brain by competitive RT-PCR.
The N-methyl-D-aspartate (NMDA)-selective subtype of ionotropic glutamate receptor is of importance in neuronal differentiation and synapse consolidation, activity-dependent forms of synaptic plasticity, and excitatory amino acid-mediated neuronal toxicity [Neurosci. Res. Program Bull. 19 (1981) 1; Lab. Invest. 68 (1993) 372]. NMDA receptors exist in vivo as tetrameric or pentameric complexes comprising proteins from two families of homologous subunits, designated NR1 and NR2(A-D) [Biochem. Biophys. Res. Commun. 185 (1992) 826]. The gene coding for the human NR1 subunit (hNR1) is composed of 21 exons, three of which (4, 20 and 21) can be differentially spliced to generate a total of eight distinct subunit variants. We detail here a competitive RT-PCR (cRT-PCR) protocol to quantify endogenous levels of hNR1 splice variants in autopsied human brain. Quantitation of each hNR1 splice variant is performed using standard curve methodology in which a known amount of synthetic ribonucleic acid competitor (internal standard) is co-amplified against total RNA. This method can be used for the quantitation of hNR1 mRNA levels in response to acute or chronic disease states, in particular in the glutamatergic-associated neuronal loss observed in Alzheimer's disease [J. Neurochem. 78 (2001) 175]. Furthermore, alterations in hNR1 mRNA expression may be reflected at the translational level, resulting in functional changes in the NMDA receptor.
Hynd
Matthew R
MR
Department of Biochemistry, University of Queensland, Brisbane 4072, Australia.
Scott
Heather L
HL
Dodd
Peter R
PR
D017398:Alternative Splicing / Q000235:genetics; D000544:Alzheimer Disease / Q000175:diagnosis / Q000235:genetics / Q000378:metabolism; D001921:Brain / Q000378:metabolism*; D001923:Brain Chemistry / Q000235:genetics*; D003001:Cloning, Molecular; D005786:Gene Expression Regulation / Q000235:genetics; D018698:Glutamic Acid / Q000378:metabolism; D006801:Humans; D009474:Neurons / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics; D021122:Protein Subunits / 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
C409466:NR1 NMDA receptor; D020033:Protein Isoforms; D021122:Protein Subunits; D012333:RNA, Messenger; D016194:Receptors, N-Methyl-D-Aspartate; D018698:Glutamic Acid