MOLECULAR BIOLOGICAL STUDIES OF CALRETININ

显微切割技术用于神经化学物质的定量 离散的脑核团已被应用于定量测量 mRNA。所述方法允许定量低丰度mRNA， 低于毫克的组织 离散核和其他区域 将脑溶解在浓硫氰酸胍中 溶液中，mRNA直接与核糖核酸探针杂交，并检测 核糖核酸酶保护试验。此方法消除了 从实体组织中分离RNA的必要性。 没有假设 关于RNA回收是必要的，因为组织标本是 在单个管中溶解、杂交和成核。同时 用总样品蛋白质检测感兴趣的mRNA， 丰富的“管家”基因（β-肌动蛋白，允许正常化， 在这些内部控制方面进行定量。我们已经确定 calretinin的水平，一种中枢神经系统神经元特异性 钙结合蛋白和β-肌动蛋白， 老鼠大脑的其他区域。Calretinin mRNA的量范围为 丘脑室旁核中的281 +/- 35 fg/mg蛋白质至2.3 大脑皮层+/- 0.5 fg/mg蛋白。 钙视网膜蛋白/β- 相同标本中肌动蛋白的比例范围为79.9 +/- 9.3%至1.3 +/- 0.1%。 我们对裂解物RNase保护的修改 分析：（1）绕过与之相关的效率低下和不确定性， 从显微解剖的脑标本中分离RNA;（2）建立 高丰度和低丰度定量检测技术 mRNA;和（3）使得能够从多个基因组中进行大量测定。 2至3天内分析脑核。

A microdissection technique for quantitation of neurochemicals in discrete brain nuclei has been applied to quantitative measurement of mRNA. The method permits quantitation of low abundance mRNA from submilligram amounts of tissue. Discrete nuclei and other regions of the brain are solubilized in concentrated guanidine thiocyanate solution, mRNA is directly hybridized with riboprobes, and detected with a ribonuclease protection assay. This method eliminates the necessity for RNA isolation from solid tissue. No assumptions regarding RNA recovery are necessary since tissue specimens are solubilized, hybridized and nucleased in a single tube. Simultaneous detection of mRNAs of interest, with total sample protein and the abundant 'housekeeping' gene (3-actin, permits normalization and quantitation in terms of these internal controls. We have determined the levels of calretinin, a central nervous system neuron-specific calcium binding protein and beta-actin in microdissected nuclei and other regions of rat brain. The quantity of calretinin mRNA ranged from 281 +/- 35 fg/mg protein in the thalamic paraventricular nucleus to 2.3 +/- 0.5 fg/mg protein for the cerebral cortex. The calretinin/beta- actin ratios in the same specimens ranged from 79.9 +/- 9.3% to 1.3 +/- 0.1%, respectively. Our modifications to the lysate RNase protection assay: (1) bypass the inefficiencies and uncertainties associated with isolating RNA from microdissected brain specimens; (2) establish this technique as quantitative for detection of high and low abundance mRNAs; and (3) enable large numbers of determinations from multiple brain nuclei to be analyzed in 2 to 3 days.