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J.Health Sci., 56(5), 527-533, 2010
-Regular Article-
Development of a SYBR Green Real-time Polymerase Chain Reaction Assay for Quantitative Detection of Human N-methyl-D-aspartate Receptors Subtype 1 Splice Variants
Susumu Ohkawara,a Toshiko Tanaka-Kagawa,b
Yoko Furukawa,b Tetsuji Nishimura,b
and Hideto Jinno*, b
aDepartment of Pharmaceutical Science, Musashino University, 1-1-20 Shinmachi, Nishitokyo-shi, Tokyo 202-8585, Japan and
bDivision of Environmental Chemistry, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan
N-methyl-D-aspartate receptors (NMDAR) belong to the ionotropic glutamate receptor subclass and are widely distributed in the vertebrate brain. Molecular cloning has revealed the existence of seven NMDAR subunits: one NMDAR1 (NR1), four different NMDAR2 (NR2A-D), and two different NMDAR3 (NR3A,B). Alternative splicing of the single NR1 gene generates eight isoforms with distinct functional properties. So far, the transcripts of the NR1 splice variants have been discriminated by Northern blot, in situ hybridization, or competitive polymerase chain reaction (PCR) methods all of which have their intrinsic limitations. In this study, we have developed a method to quantify the mRNAs of the NR1 splice variants by real-time PCR with the double-stranded DNA-binding dye SYBR Green I. The implementation of this assay will allow a better understanding of the regulatory mechanisms of the NR1 splice variants, and hence, their role in neuronal disease pathogenesis.
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