Characterization of rat rostral raphe primary cultures: Multiplex quantification of serotonergic markers
Characterization of rat rostral raphe primary cultures: Multiplex quantification of serotonergic markers
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DOI：
10.1016/j.jneumeth.2007.04.002
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发表时间：
2007-08-15
影响因子：
3
通讯作者：
Albert, Paul R.
中科院分区：
文献类型：
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作者：
Czesak, Margaret;Bums, Ariel M.;Albert, Paul R.
先前建立中缝培养物的报告通常产生不到 1% 的血清素 (5-HT) 阳性神经元，并且对于转录研究来说是不切实际的。在这项研究中，我们建立了富含 5-HT 神经元的原代培养物，并量化了表达血清素能和非血清素能标记物的细胞比例。我们还展示了使用多重实时 PCR 技术来测量其中一些标记物的 RNA 相对量的可行性。将源自E1 3-15大鼠胚胎的中缝嘴细胞培养7天，并通过定量免疫荧光和蛋白质印迹分析进行分析。在这些培养物中，-8% 的神经元对血清素能标记物（5-HT 或色氨酸羟化酶 (TPH)）呈免疫阳性。标记为 GFAP（神经胶质标记物）、酪氨酸羟化酶（儿茶酚胺能）和 GAD65/67（GABA 能）的细胞百分比分别为 5%、1% 和 54%。转录因子 REST/NRSF 和 Deaf-1 分别存在于 9% 和 98% 的细胞中。对与作为对照的 GAPDH RNA 配对的 TPH2、5-HT1A 受体或 Deaf-1 RNA 进行多重定量 RT-PCR (Q-PCR) 分析。使用这种方法，在 200 倍稀释浓度范围内获得了每种 RNA 的标准曲线，r2 值 > 0.99。 Q-PCR 测定的相对丰度与血清素能中缝细胞中 TPH2 > Deaf-1 > 5-HT1A 受体 RNA 的表达一致。培养物之间 TPH2 RNA 水平的标准误差为
Previous reports establishing raphe cultures typically yield less than 1% serotonin (5-HT)-positive neurons and are impractical for transcriptional studies. In this study, we have established primary cultures enriched in 5-HT neurons and quantified the proportion of cells expressing serotonergic and non-serotonergic markers. We have also shown the feasibility of using the multiplex real-time PCR technique to measure the relative amounts of RNA for some of these markers. Rostral raphe cells derived from El 3-15 rat embryos were cultured for 7 days and analyzed by quantitative immunofluorescence and western blot analysis. In these cultures, -8% of neurons were immunopositive for serotonergic markers (5-HT or tryptophan hydroxylase (TPH)). The percentage of cells labeled for GFAP (glial marker), tyrosine hydroxylase (catecholaminergic), and GAD65/67 (GABAergic) was 5, 1, and 54%, respectively. Transcription factors REST/NRSF and Deaf-1 were present in 9 and 98% of cells, respectively. Multiplex quantitative RT-PCR (Q-PCR) analysis was done for TPH2, 5-HT1A receptor or Deaf-1 RNAs paired with GAPDH RNA as control. Using this approach, standard curves for each RNA were obtained over 200-fold concentration range of dilution with r2 values >0.99. The relative abundances determined by Q-PCR are consistent with the expression of TPH2 > Deaf-1 > 5-HT1A receptor RNA in serotonergic raphe cells. The standard error of TPH2 RNA levels between cultures was