Gautreau A, Kerdelhué B
Laboratoire de Neuroendocrinologie, CNRS URA 1310, Faculté de Pharmacie, Paris, France.
Brain Res Brain Res Protoc. 1998 Jan;2(2):133-40. doi: 10.1016/s1385-299x(97)00036-6.
Tachykinins form a family of peptides with neurotransmitter/neuromodulator function. Four tachykinins, substance P, neurokinin A, neuropeptide gamma and neuropeptide K, are encoded by the same PreProTachykinin (PPT) gene. Alternatively spliced mRNAs encode different combinations of these peptides (Brown, E.R., Harlan, R.E., Krause, J.E., Gonadal steroid regulation of substance P (SP) and SP-encoding mRNA in the rat anterior pituitary and hypothalamus, Endocrinology, 126 (1990) 330-340; Krause, J.E., Chirgwin, J.M., Carter, M.S., Xu, Z.S., Hershey, A.D., Three rat preprotachykinin mRNAs encode the neuropeptides substance P and neurokinin A, Proc. Natl. Acad. Sci. USA, 84 (1987) 881-885). The proportion of PPT mRNAs varies from tissue to tissue (Carter, M.S., Krause, J.E., Structure, expression, and some regulatory mechanisms of the rat preprotachykinin gene encoding substance P, neurokinin A, neuropeptide K, and neuropeptide gamma, J. Neurosci., 10 (1990) 2203-2214), and within the rat hypothalamus according to the estrous cycle-related hormonal status (Gautreau, A., Duval, P., Kerdelhué, B., Variations in substance P-encoding preprotachykinin and substance P receptor NK-1 mRNA transcripts in the rat hypothalamus throughout the estrous cycle: a correlation between amounts of beta-preprotachykinin and NK-1 mRNA, Mol. Brain Res., (1997) in press). Tachykinin receptors as well as tachykinins are regulated at the mRNA level. A fully quantitative method is needed to deal with the complex physiological regulation of the tachykinin system. Here, we describe an RNase protection assay that allows the simultaneous quantitation of alternatively spliced PPT mRNAs, Substance P receptor NK-1 mRNA, and glyceraldehyde-3-phosphodehydrogenase (GAPDH) mRNA as an internal control, in the rat hypothalamus. The advantages of this method are its high sensitivity (0.1 pg) and a wide range of linearity (more than 3 orders of magnitude). Moreover, this protocol provides guidelines to set up a quantitative multiprobe RNase protection assay for other genes.
速激肽形成了一个具有神经递质/神经调质功能的肽家族。四种速激肽，即P物质、神经激肽A、神经肽γ和神经肽K，由同一个前速激肽原（PPT）基因编码。可变剪接的mRNA编码这些肽的不同组合（Brown, E.R., Harlan, R.E., Krause, J.E., 大鼠垂体前叶和下丘脑P物质（SP）及编码SP的mRNA的性腺类固醇调节，《内分泌学》，126 (1990) 330 - 340；Krause, J.E., Chirgwin, J.M., Carter, M.S., Xu, Z.S., Hershey, A.D., 三种大鼠前速激肽原mRNA编码神经肽P物质和神经激肽A，《美国国家科学院院刊》，84 (1987) 881 - 885）。PPT mRNA的比例因组织而异（Carter, M.S., Krause, J.E., 编码P物质、神经激肽A、神经肽K和神经肽γ的大鼠前速激肽原基因的结构、表达及一些调节机制，《神经科学杂志》，10 (1990) 2203 - 2214），并且在大鼠下丘脑内，其比例会根据发情周期相关的激素状态而变化（Gautreau, A., Duval, P., Kerdelhué, B., 大鼠下丘脑在整个发情周期中编码P物质的前速激肽原和P物质受体NK - 1 mRNA转录本的变化：β - 前速激肽原和NK - 1 mRNA量之间的相关性，《分子脑研究》，(1997) 即将发表）。速激肽受体以及速激肽在mRNA水平上受到调节。需要一种完全定量的方法来处理速激肽系统复杂的生理调节。在此，我们描述了一种核糖核酸酶保护分析方法，该方法能够同时定量大鼠下丘脑中可变剪接的PPT mRNA以及P物质受体NK - 1 mRNA，并将甘油醛 - 3 - 磷酸脱氢酶（GAPDH）mRNA作为内参。该方法的优点是灵敏度高（0.1 pg）且线性范围广（超过3个数量级）。此外，本方案为针对其他基因建立定量多探针核糖核酸酶保护分析提供了指导原则。
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