Navigation Links
Gene-Specific Primers for NMDA Receptor Subunits

Additional tools for neuroscience researchers


Robert Buchner Peter L. Pingerelli Michael D. Browning Mary Ellen Simcox
Stratagene Cloning Systems, Inc.


Stratagene is now offering gene-specific primer sets for the rat and human NMDA receptor. In this article, we describe using these primers in reverse transcriptase-mediated polymerase chain reaction (RT-PCR) assays to show expression of NMDA subunits with Stratagenes rat cDNA, human cDNA and hNT neuron cDNA libraries as templates. By using these primers, researchers will be able to study rat and human NMDAR subunit-specific expression in a variety of different cell types and regions of the brain.

The ion channels activated by glutamate are typically divided into two classes. The ion channels that are sensitive to N-methyl-D-aspartate (NMDA) are designated NMDA receptors (NMDAR), while those activated by kainate and a-amino-3-hydroxy-5-methyl-4-isoxalone propionic acid (AMPA) are known as kainate/AMPA receptors (K/AMPAR). The NMDAR assumes a critical role in long-term potentiation (LTP), a putative cellular substrate of memory.1 This receptor has also been linked to neuronal development and has been implicated in several disorders of the central nervous system, including epilepsy and ischemic neuronal cell death.

The rat NMDAR1 (NR1) was the first subunit of the NMDAR to be cloned.2 The NR1 protein can form NMDA-activated ion channels when expressed in Xenopus oocytes, but the currents in such channels are much weaker than those seen in situ. Channels with more relevant physiological characteristics are produced when the NR1 subunit is combined with one or more of the NMDAR2 (NR2A-D) subunits.3 In addition, there are a number of different splice variants of the NR1.4 Significant functional diversity has be en seen in recombinant systems with heterologous coexpression of various NR2 subunits and/or NR1 splice variants.5 However, little is known about the expression of the NMDAR in situ.

NMDA Receptor Subunit

Expected PCR Product Size (bp)

NR1, Rat; flanking C1 exon

C1+, 214 C1, 103

NR1, Rat: flanking N1 exon

N1+, 235 N1,172

NR2A, Rat

207

NR2B, Rat

183

NR1, Human

782

Table 1

figure 1

RT-PCR assays with gene-specific primer sets can be used to study gene expression of the NMDA receptor subunits.6 Stratagenes cDNA library templates and amplification primer sets6 for rat NMDA subuni ts were used for RT-PCR reactions to identify cell populations expressing these specific NMDA receptor subunit genes. Briefly, RT-PCR was conducted using species-specific primer sets for various NMDA receptor subunits (table 1) with cDNA constructed from whole rat brain (figure 1). (Data from reactions using human brain and human hNT cells as templates are not shown). PCR products resulting from amplification of rat NMDA receptor subunit genes were electrophoresed on an agarose gel (figure 1), and their sizes were visualized by ethidium bromide staining. Amplification products with the gene-specific primers for the NMDA receptor subunits resulted in products with correct sizes for all cDNAs tested. For NR1, amplification using primers flanking the C1 or N1 splice-variant exons from rat resulted in two products of different sizes, corresponding to mRNAs either containing or lacking these exons.

Conclusions

Investigators can now use the extremely sensitive RT-PCR method to study expression of NMDA receptor subunit genes. Stratagenes RT-PCR primers for rat and human NMDA receptor genes have been tested using cDNA templates derived from whole brain or cultured hNT cells. These new RT-PCR primers will allow further studies of the subunit composition of NMDA receptors.

REFERENCES
  1. Collingridge, G.L., et al. (1983) J. Physiol. (Lond.) 335: 33-46.

  2. Moriyoshi, K., et al. (1991) Nature 354: 31-37.

  3. Monyer, H., et al. (1992) Science 256: 1217-1221.

  4. Laurie, D.J., Seeburg, P.H. (1994) J. Neurosci. 14: 3180-3194.

  5. Monyer, H., et al. (1994) Neuron 12: 529-540.

  6. Sheng, M., et al. (1994) Nature 368: 144-147.


'"/>

Source:


Page: All 1 2 3 4

Related biology technology :

1. Control RT-PCR Primers for Human Gene Transcripts with Varying Abundance
2. RT-PCR Primers for the Study of Apoptosis
3. New PCR Primers for Human Diseases
4. Amplification of Epstein-Barr Virus Exon C with Difficult Primers Using Eppendorf Mastercycler gradient
5. Antibodies for Studying NMDA Receptor Protein Expression and Synapse-Specific Immunolabeling
6. Detecting Gastrin-Releasing Peptide Receptor by in situ PCR on Archived Tissue
7. pFB-ERV: Retroviral Delivery of the Ecdysone Receptor Proteins
8. TKB1 Cells Identify Receptor Tyrosine Kinase Interacting Proteins
9. Quantification of the Purinergic Receptor P2X3 using ICAT and Orthogonal 2D LC-MS/MS with an Ion Trap Mass Spectrometer
10. Isolation of Low Molecular Weight Digestion Products of the Human Platelet Thromboxane A2 Receptor, Rev A
11. Performance of Ad-A-Gene EGFP-Glucocorticoid Receptor (EGFP-GCCR): an adenoviral vector gene delivery system
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:12/2/2016)... ... 01, 2016 , ... ACEA Biosciences, Inc. announced today that it will be ... at the World Conference on Lung Cancer 2016, taking place in Vienna, Austria December ... clinical trials for AC0010 in patients with advanced non-small cell lung cancer harboring the ...
(Date:12/2/2016)... ... 2016 , ... DrugDev believes the only way to achieve real ... All three tenets were on display at the 2nd Annual DrugDev User Summit (hosted ... CRO and site organizations to discuss innovation and the future of clinical research. ...
(Date:12/2/2016)... PUNE, India , December 2, 2016 ... Billion by 2021, growing at a CAGR of 7.3% during the ... segment while hospitals and diagnostic laboratories segment accounted for the largest ... ... Complete report on global immunohistochemistry (IHC) market spread across 225 ...
(Date:11/30/2016)... 2016 /PRNewswire/ - Portage Biotech Inc. ("Portage" or "the ... excited to announce the formation of EyGen, Ltd. ... ophthalmology assets through proof of concept. EyGen,s lead ... Portage Pharmaceuticals Limited and being developed for topical ... anterior segment diseases. This agent has the potential ...
Breaking Biology Technology:
(Date:11/24/2016)... -- Cercacor today introduced Ember TM Sport Premium ... measure hemoglobin, Oxygen Content, Oxygen Saturation, Perfusion Index, ... approximately 30 seconds. Smaller than a smartphone, using only ... key data about their bodies to help monitor these ... Hemoglobin carries oxygen to muscles. When hemoglobin and ...
(Date:11/17/2016)... 17, 2016 Global Market Watch: Primarily ... Banks, Population-Based Banks and Academics) market is to witness a ... Biobanks shows the highest Compounded Annual Growth Rate (CAGR) of ... during the analysis period 2014-2020. North America ... followed by Europe at 9.56% respectively. ...
(Date:11/14/2016)... 14, 2016  xG Technology, Inc. ("xG" or the ... wireless communications for use in challenging operating environments, announced ... 2016. Management will hold a conference call to discuss ... Eastern Time (details below). Key Recent Accomplishments ... million binding agreement to acquire Vislink Communication Systems. The ...
Breaking Biology News(10 mins):