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Quantitation of NMDA receptor NR2 mRNA transcripts in human brain by competitive RT-PCR

Authors
Journal
Brain Research Protocols
1385-299X
Publisher
Elsevier
Publication Date
Volume
11
Issue
1
Identifiers
DOI: 10.1016/s1385-299x(03)00017-5
Keywords
  • Nmda Receptor
  • Nr2 Subtypes
  • Competitive Rt-Pcr
  • Gene Expression
  • Mrna
Disciplines
  • Biology

Abstract

Abstract 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.

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