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 (GFRα-2) is the preferred receptor for NTN. In addition to the known full-length isoform (GFRα-2a), we have previously reported the isolation of two novel alternatively spliced isoforms (GFRα-2b and GFRα-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 (GFRα-2a, GFRα-2b and GFRα-2c). Of the three isoforms, GFRα-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 GFRα-2 isoforms and can be extended to the quantitation of other alternatively spliced isoforms.