Wong Y W, Sia G M, Too H P
Department of Biochemistry, National University of Singapore, Lower Kent Ridge Crescent, Singapore 119260, Singapore.
Neurosci Lett. 2002 Mar 8;320(3):141-5. doi: 10.1016/s0304-3940(01)02282-0.
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.
神经营养因子（NTN）属于胶质细胞源性神经营养因子（GDNF）家族的生长因子。NTN和GDNF在体外和体内均已显示出能有效防止多巴胺能神经元的退化。GDNF家族受体α2（GFRα-2）是NTN的首选受体。除了已知的全长异构体（GFRα-2a）外，我们之前还报道了两种新的可变剪接异构体（GFRα-2b和GFRα-2c）的分离。所有这三种异构体在所有检测的哺乳动物组织中均有表达，包括人类胎儿大脑。然而，这些异构体的表达水平尚未进行定量。在本报告中，我们开发了一种使用SYBR Green I的实时聚合酶链反应（PCR）检测方法，以检测三种剪接变体（GFRα-2a、GFRα-2b和GFRα-2c）的表达水平。在这三种异构体中，发现GFRα-2a是在整个小鼠大脑中表达最丰富的受体。本报告中开发的使用SYBR Green I的实时PCR检测方法可用于明确量化GFRα-2异构体的表达水平，并可扩展到其他可变剪接异构体的定量分析。
