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

单细胞水平脑神经元表达的谷氨酸受体亚基 mRNA 的绝对定量

基本信息

批准号：
07558109
负责人：
OZAWA Seiji
金额：
$ 6.34万
依托单位：
Gunma University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

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

我们先前已经表明，可以使用斑块夹夹转录 - colymerase链反应（Patch-Clamp RT-PCR）技术在单细胞水平上分析天然AMPA型谷氨酸受体的功能特性的分子基础。但是，先前的方法不允许对单个细胞中表达的mRNA量进行定量估计。该项目的目的是建立一种在单个细胞水平上在脑神经元中表达的GLUR2 AMPA受体亚基mRNA的绝对定量方法。结果分为以下两个部分，首先是定量方法的建立，其次是单神经元分析方法的应用。1）量化glur2 mRNA在大鼠前脑和小脑中的glur2 mRNA量的量化估计方法是基于基于易生成的转录gllna的ct rya crom from the the Artra crom from the Artra crom from the terna crom from the terna crom from the ternice crom from 2 MRNNA。更多的NA，其中进行了2098位从C到G的单基础交换，以破坏野生型的BSP1286I，并创建一个独特的Stui位点，作为内标。转化为cDNA和PCR后，可以使用适当的限制酶消化扩展的DNA产物，以区分从内源性靶标mRNA和凝胶电噬菌体源自内标RNA的DNA。用于PCR的反义引物用 ^<32> P标记，并且消化的PCR产物的量通过BAS2000（Fuji膜）量化。 It was estimated by this method that the forebrain and cerebellum of rats containing 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 neuroThe patch-clamp RT-PCR technique using the internal standard was applied to estimate the amounts of GluR2 mRNA expressed在由大鼠胚胎制备的原代培养物中的单个金字塔神经元中。在测试的11个神经元中，GLUR2 mRNA的副本数量从100到3000不等。平均数为1015*317（平均*SE）。较少的