Real-time quantitative RT-PCR for low-abundance transcripts in the inner ear: analysis of neurotrophic factor expression
Real-time quantitative RT-PCR for low-abundance transcripts in the inner ear: analysis of neurotrophic factor expression
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DOI：
10.1016/s0378-5955(03)00298-3
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发表时间：
2003-11-01
期刊：
影响因子：
2.8
通讯作者：
Corfas, G
中科院分区：
文献类型：
--
作者：
Stankovic, KM;Corfas, G
实时定量逆转录-PCR是一种高灵敏度的技术，可以高通量定量基因表达。将该技术应用于内耳可能非常重要，但并不简单，因为组织采集可能具有挑战性，来自单个内耳的RNA产量低，并且从缺乏RNA合成cDNA可能效率低下。为了克服这些挑战，我们测试了许多参数和试剂，并开发了一种方法来可靠地定量低丰度转录物中的微小变化。使用这种技术，我们证明了神经营养因子神经营养素3（NT-3），脑源性神经营养因子（BDNF）和胶质细胞系源性神经营养因子（GDNF）的存在和定量。在出生后小鼠内耳的耳蜗和前庭终器中（P26）。我们发现，在测试的因素中，BDNF是耳蜗和前庭终末器官之间唯一的差异表达，在前庭终末器官中的丰度是23.4 +/- 0.3倍。在耳蜗内，GDNF基因表达是NT-3表达的4.9 +/- 0.2倍。在联合前庭终末器官中，BDNF的表达是NT-3表达的43.0 +/- 1.5倍。我们的研究结果表明，神经营养因子继续发挥作用，在出生后的内耳，除了他们以前显示的重要作用在发展过程中。(C)2003 Elsevier B. V.保留所有权利。
Real-time quantitative reverse transcription-PCR is a highly sensitive technology that allows high throughput quantification of gene expression. Application of this technique to the inner ear is potentially very important, but is not straightforward because tissue harvesting can be challenging, RNA yield from individual inner ears is low, and cDNA synthesis from scant RNA can be inefficient. To overcome these challenges, we tested many parameters and reagents, and developed an approach to reliably quantitate small changes in low-abundance transcripts. Using this technique we demonstrate the presence and quantify amounts of the neurotrophic factors neurotrophin 3 (NT-3), brain-derived neurotrophic factor (BDNF) and glial cell-line-derived neurotrophic factor (GDNF),. in the cochlea and vestibular end organs of postnatal murine inner ear (P26). We show that out of the factors tested, BDNF is the only one differentially expressed between the cochlea and vestibular end organs, being 23.4 +/- 0.3 times more abundant in the vestibular end organs. Within the cochlea, GDNF gene expression is 4.9 +/- 0.2 times greater than NT-3 expression. Within the combined vestibular end organs, BDNF expression is 43.0 +/- 1.5 times greater than NT-3 expression. Our results suggest that neurotrophic factors continue to play a role in the postnatal inner ear, in addition to their previously shown essential role during development. (C) 2003 Elsevier B.V. All rights reserved.