Absolute quantification of mRNA of glutamate receptor subunit expressed in brain neurons at the single cell level

我们先前已经证明，利用膜片钳反转录-聚合酶链式反应(Patch-Chare RT-PCR)技术可以在单细胞水平上分析天然AMPA型谷氨酸受体功能特性的分子基础。然而，以前的方法不能定量估计单个细胞中表达的信使核糖核酸的数量。本项目旨在建立一种在单个细胞水平上绝对定量检测GluR2 AMPA受体亚单位在脑神经元中表达的方法。结果分为两个部分，第一部分是定量方法的建立，第二部分是单神经元分析方法的应用1)大鼠前脑和小脑中GluR2mRNAs的数量的定量估计该方法是基于体外产生的转录物的共扩增，该转录物与从突变的CD…体外转录的靶向GluR2mRNA.RNA不同More NA作为内标，在2098位进行C到G的单碱基交换，以破坏野生型Bsp1286I，并创建一个独特的StuI位点。将扩增的DNA产物转化为cDNA和PCR后，用适当的限制性内切酶进行酶切，用凝胶电泳法区分内源性目的基因和内标基因。用~(32)&gt；P标记反义引物，用BAS2000(富士胶片)对扩增产物进行定量。用该方法估计大鼠前脑和小脑分别含有1.0×10~(-5)和1.6×10~(-4)拷贝的GluR2 m RNA/ng。2)单个培养的海马神经元GluR2 m RNA表达的定量估计。在被测试的11个神经元中，GluR2基因的拷贝数从100到3000不等。平均数为1015×317(平均值*SE)。较少

We have previously shown that the molecular basis of functional properties of native AMPA-type glutamate receptors can be analyzed at the single cell level by using the patch-clamp reverse transcription-polymerase chain reaction (Patch-clamp RT-PCR) technique. However, the previous method did not allow quantitative estimation of the amounts of mRNA expressed in a single cell. This project aimed to establish a method for absolute quantification of mRNA of GluR2 AMPA receptor subunit expressed in brain neurons at the single, cell level. The results are divided into the following two parts, firstly the establishment of the method for quantification and secondly the application of the method for single-neuron analysis.1) Quantitative estimation of the amounts of GluR2 mRNAs in rat forebrain and cerebellumThe method was based on the co-amplification of an in vitro generated transcript differing by a single base change from the targeted GluR2 mRNA.RNA transcribed in vitro from the mutated cD … More NA, in which a single-base exchange from C to G at position 2098 was done to destroy Bsp1286I of the wild-type and to create a unique StuI site, served as internal standard. After conversion to cDNA and PCR, the amplified DNA products could be digested with the appropriate restriction enzymes to discriminate between DNA derived from endogenous target mRNA and DNA derived from internal standard RNA by gel-electrophoresis. The antisense primer used for PCR was labelled by ^<32>P and the amount of the digested PCR product was quatified by BAS2000 (Fuji Film). It was estimated by this method that the forebrain and cerebellum of rats contain 1.0x 10^5 and 1.6 x 10^4 copies of GluR2 mRNA/ng RNA, respectively.2) Quantitative estimation of the amounts of GluR2 mRNAs expressed in a single cultured hippocampal neuronThe patch-clamp RT-PCR technique using the internal standard was applied to estimate the amounts of GluR2 mRNA expressed in a single pyramidal neurons in primary cultures prepared from rat embryos. In 11 neurons tested, the number of copies of GluR2 mRNA varied from 100 to 3000. The mean number was 1015*317 (mean*SE). Less