Quantification of mouse glial cell-line derived neurotrophic factor family receptor alpha 2 alternatively spliced isoforms by real time detection PCR using SYBR Green I.
Neurturin (NTN) belongs to the glial cell-line derived neurotrophic factor (GDNF) family of growth factors. Both NTN and GDNF have been shown to potently prevent the degeneration of dopaminergic neuron in vitro and in vivo. The GDNF family receptor alpha 2 (GFR alpha-2) is the preferred receptor for NTN. In addition to the known full-length isoform (GFR alpha-2a), we have previously reported the isolation of two novel alternatively spliced isoforms (GFR alpha-2b and GFR alpha-2c). All three isoforms are expressed in all mammalian tissues examined, including human fetal brain. However, the expression levels of these isoforms have yet to be quantified. In this report, we have developed a real time polymerase chain reaction (PCR) detection method using SYBR Green I to detect the expression levels of the three splice variants (GFR alpha-2a, GFR alpha-2b and GFR alpha-2c). Of the three isoforms, GFR alpha-2a was found to be the most abundant receptor expressed in the whole murine brain. The real time PCR detection method using SYBR Green I developed in this report can be used to unambiguously quantitate expression levels of the GFR alpha-2 isoforms and can be extended to the quantitation of other alternatively spliced isoforms.
Wong
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YW
Department of Biochemistry, National University of Singapore, Lower Kent Ridge Crescent, Singapore 119260, Singapore.
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D017398:Alternative Splicing / Q000235:genetics*; D000818:Animals; D052160:Benzothiazoles; D001921:Brain / Q000378:metabolism; D001923:Brain Chemistry / Q000235:genetics*; D017931:DNA Primers; D003959:Diamines; D029721:Drosophila Proteins; D005456:Fluorescent Dyes; D051097:Glial Cell Line-Derived Neurotrophic Factor Receptors; D051379:Mice; D009154:Mutation / Q000235:genetics; D009414:Nerve Growth Factors / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism; D051101:Neurturin; D015345:Oligonucleotide Probes; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods*; D020033:Protein Isoforms / Q000235:genetics / Q000302:isolation & purification; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000302:isolation & purification*; D051096:Proto-Oncogene Proteins c-ret; D011804:Quinolines; D012333:RNA, Messenger / Q000378:metabolism; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics / Q000302:isolation & purification*; D015203:Reproducibility of Results
D052160:Benzothiazoles; D017931:DNA Primers; D003959:Diamines; D029721:Drosophila Proteins; D005456:Fluorescent Dyes; C494991:Gfra2 protein, mouse; D051097:Glial Cell Line-Derived Neurotrophic Factor Receptors; D009414:Nerve Growth Factors; D051101:Neurturin; C495056:Nrtn protein, mouse; D015345:Oligonucleotide Probes; D009930:Organic Chemicals; D020033:Protein Isoforms; D011518:Proto-Oncogene Proteins; D011804:Quinolines; D012333:RNA, Messenger; C098022:SYBR Green I; D051096:Proto-Oncogene Proteins c-ret; D020794:Receptor Protein-Tyrosine Kinases; C416649:Ret protein, Drosophila