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首页> 外文期刊>Journal of Neuroscience Methods >Analysis of tyrosine hydroxylase gene transcription using an intron specific probe.
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Analysis of tyrosine hydroxylase gene transcription using an intron specific probe.

机译：使用内含子特异性探针分析酪氨酸羟化酶基因转录。

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摘要

The nuclear run-on assay is the most commonly used technique to determine transcription rates of specific genes such as tyrosine hydroxylase. Its application to studies in the nervous system is problematic, however, as a result of limitations in sensitivity and the loss of anatomical integrity. We observed that the relative levels of tyrosine hydroxylase intron 2-containing RNA using a ribonuclease protection assay in the adrenal medulla changed in response to pharmacological treatments consistently with changes shown by the nuclear run-on assay. Our results indicate that measures of tyrosine hydroxylase primary transcript levels offer an alternative to the nuclear run-on assay and validate the application of intron-specific in situ hybridization as a means of assessing the relative transcriptional activity of the tyrosine hydroxylase gene. Similar quantitative results were obtained using intron-specific in situ hybridization with oligonucleotide probes specific for rat tyrosine hydroxylase intron 2. Furthermore, we observed that intron-specific in situ hybridization could be used to measure tyrosine hydroxylase transcription rates in the locus coeruleus, providing resolution at the level of single neurons. Thus, measuring the levels of tyrosine hydroxylase intron 2 provides a sensitive measure of tyrosine hydroxylase transcription rate that can be applied to the study of brain catecholaminergic neurons.
机译：核运行分析是确定特定基因（例如酪氨酸羟化酶）转录速率的最常用技术。然而，由于敏感性限制和解剖学完整性的丧失，其在神经系统研究中的应用存在问题。我们观察到，在肾上腺髓质中使用核糖核酸酶保护测定法，含有酪氨酸羟化酶内含子2的RNA的相对水平响应于药理学治疗而发生了变化，与核运行测定法所显示的变化一致。我们的结果表明，酪氨酸羟化酶一级转录水平的测量为核运行分析提供了一种替代方法，并验证了内含子特异性原位杂交的应用作为评估酪氨酸羟化酶基因相对转录活性的一种手段。使用内含子特异性原位杂交与对大鼠酪氨酸羟化酶内含子2特异的寡核苷酸探针获得相似的定量结果。此外，我们观察到内含子特异性原位杂交可用于测量蓝斑中酪氨酸羟化酶的转录速率，从而提供分辨率在单个神经元的水平。因此，测量酪氨酸羟化酶内含子2的水平提供了一种敏感的酪氨酸羟化酶转录速率测量方法，可用于研究脑儿茶酚胺能神经元。

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