Clark M S, Russo A F
Molecular Biology Program, University of Iowa, Iowa City, IA 52242, USA.
Brain Res Brain Res Protoc. 1998 Jun;2(4):273-85. doi: 10.1016/s1385-299x(98)00004-x.
Tryptophan hydroxylase (TPH) is the rate limiting enzyme in serotonin biosynthesis [D.G. Grahame-Smith, Tryptophan hydroxylation in brain, Biochem. Biophys. Res. Commun. 16 (1964) 586-592 [19]]. As such, the TPH gene is a likely target for modulation of serotonergic function, which has been associated with several psychiatric disorders [E.C. Azmitia, P.M. Whitaker-Azmitia, Awakening the sleeping giant: anatomy and plasticity of the brain serotonergic system, J. Clin. Psychiatry 52 (12, Suppl.) (1991) 4-16 [1]; R.P. Hart, R. Yang, L.A. Riley., T.L. Green, Post-transcriptional control of tryptophan hydroxylase gene expression in rat brain stem and pineal gland, Mol. Cell. Neurosci. 2 (1991) 71-77 [20]; M.J. Owens, C.B. Numeroff, Role of serotonin in the pathophysiology of depression: focus on the serotonin transporter, Clin. Chem. 40 (1994) 288-295 [24]]. Unfortunately, it has been technically difficult to measure TPH mRNA levels in central serotonergic neurons due to its low levels. For example, detection with ribonuclease protection assays requires pooling of 5-10 dissected brainstems [M.C. Darmon, B. Guibert, V. Leviel, M. Ehret, M. Maitre, J. Mallet, Sequence of two mRNAs encoding active rat tryptophan hydroxylase, J. Neurochem. 51 (1988) 312-316 [15]; B.L. Jacobs, E.C. Azmitia, Structure and function of the brain serotonin system, Physiol. Rev. 72 (1992) 165-229 [21]]. This protocol describes the use of competitive RT-PCR to measure TPH mRNA levels from rat brain. First described in 1988, competitive RT-PCR has become an accepted method of measuring RNA abundance [M. Clementi, S. Menzo, P. Bagnarelli, A. Manzin, A. Valenza, P.E. Varaldo, Quantitative PCR and RT-PCR in virology, PCR Methods Appl. 2 (1994) 191-196 [12]; N.C.P. Cross, Quantitative PCR techniques and applications, Br. J. Haematol. 89 (1995) 693-697 [14]; K.P. Foley, M.W. Leonard, J.D. Engel, Quantitation of RNA using the polymerase chain reaction, Trends Genet. 9 (1993) 380-385 [17]; P.D. Siebert, J.W. Larrick, Competitive PCR, Nature 359 (1992) 558 [27]]. Competitive RT-PCR uses co-amplification with a known quantity of an in vitro transcribed RNA which amplifies using the same primers and thus competes for reactants with the product of interest. As the two products amplify with the same efficiency, the relative abundance of the two amplification products remains constant, and thus can be used to determine initial tissue TPH mRNA levels [G. Gilliland, S. Perrin, K. Blanchard, H.F. Bunn, Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction, Proc. Natl. Acad. Sci. U.S.A. 87 (1990) 2725-2729 [18]; A.M. Wang, M. V. Doyle, D.F. Mark, Quantitation of mRNA by the polymerase chain reaction, Proc. Natl. Acad. Sci. U.S.A. 86 (1989) 9717-9721 [31]]. We first demonstrate equivalent results between RNA slot blots and competitive RT-PCR using the CA77 thyroid C cell line [M.S. Clark, A. F. Russo, Tissue-specific glucocorticoid regulation of tryptophan hydroxylase mRNA levels, Mol. Brain Res. 48 (1997) 346-354 [9]]. We then describe the use of competitive RT-PCR to measure TPH mRNA levels in RNA isolated from rat brain poly-A+ RNA.
色氨酸羟化酶（TPH）是5-羟色胺生物合成中的限速酶[D.G.格雷厄姆 - 史密斯，《脑中色氨酸的羟化》，《生物化学与生物物理研究通讯》16（1964年）586 - 592 [19]]。因此，TPH基因可能是调节5-羟色胺能功能的靶点，而5-羟色胺能功能与多种精神疾病相关[E.C.阿兹米蒂亚，P.M.惠特克 - 阿兹米蒂亚，《唤醒沉睡的巨人：脑5-羟色胺能系统的解剖与可塑性》，《临床精神病学杂志》52（12，增刊）（1991年）4 - 16 [1]；R.P.哈特，R.杨，L.A.赖利，T.L.格林，《大鼠脑干和松果体中色氨酸羟化酶基因表达的转录后调控》，《分子与细胞神经科学》2（1991年）71 - 77 [20]；M.J.欧文斯，C.B.努梅罗夫，《5-羟色胺在抑郁症病理生理学中的作用：聚焦于5-羟色胺转运体》，《临床化学》40（1994年）288 - 295 [24]]。不幸的是，由于其水平较低，在中枢5-羟色胺能神经元中测量TPH mRNA水平在技术上存在困难。例如，用核糖核酸酶保护分析法进行检测需要汇集5 - 10个解剖的脑干[M.C.达蒙，B.吉贝尔，V.勒维耶，M.埃雷，M.梅特尔，J.马莱，《两种编码活性大鼠色氨酸羟化酶的mRNA序列》，《神经化学杂志》51（1988年）312 - 316 [15]；B.L.雅各布斯，E.C.阿兹米蒂亚，《脑5-羟色胺系统的结构与功能》，《生理学评论》72（1992年）165 - 229 [21]]。本方案描述了使用竞争性逆转录 - 聚合酶链反应（RT-PCR）来测量大鼠脑中TPH mRNA水平。竞争性RT-PCR于1988年首次被描述，已成为一种公认的测量RNA丰度的方法[M.克莱门蒂，S.门佐，P.巴尼亚雷利，A.曼津，A.瓦伦扎，P.E.瓦拉尔多，《病毒学中的定量PCR和RT-PCR》，《PCR方法与应用》2（1994年）191 - 196 [12]；N.C.P.克罗斯，《定量PCR技术及其应用》，《英国血液学杂志》89（1995年）693 - 697 [14]；K.P.福利，M.W.伦纳德，J.D.恩格尔，《使用聚合酶链反应定量RNA》，《遗传学趋势》9（1993年）380 - 385 [17]；P.D.西伯特，J.W.拉里克，《竞争性PCR》，《自然》359（1992年）558 [27]]。竞争性RT-PCR使用与已知量的体外转录RNA共同扩增，该体外转录RNA使用相同的引物进行扩增，因此与感兴趣的产物竞争反应物。由于两种产物以相同的效率扩增，两种扩增产物的相对丰度保持恒定，因此可用于确定初始组织TPH mRNA水平[G.吉利兰，S.佩林，K.布兰查德，H.F.邦恩，《细胞因子mRNA和DNA的分析：通过竞争性聚合酶链反应进行检测和定量》，《美国国家科学院院刊》87（1990年）2725 - 2729 [18]；A.M.王，M.V.多伊尔，D.F.马克，《通过聚合酶链反应定量mRNA》，《美国国家科学院院刊》86（1989年）9717 - 9721 [31]]。我们首先使用CA77甲状腺C细胞系证明RNA斑点印迹法和竞争性RT-PCR之间的等效结果[M.S.克拉克，A.F.鲁索，《色氨酸羟化酶mRNA水平的组织特异性糖皮质激素调节》，《分子脑研究》48（1997年）346 - 354 [9]]。然后我们描述使用竞争性RT-PCR来测量从大鼠脑多聚腺苷酸（poly - A+）RNA中分离的RNA中的TPH mRNA水平。
