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

我们先前表明，通过使用贴片钳反转录 - 聚合酶链反应（Patch-Clamp RT-PCR）技术，天然AMPA型谷氨酸受体的分子基础可以在EVEL中进行。在单个细胞中表达的mRNA量或在单个细胞水平上表达的Glur2 Ampa受体亚基mRNA的绝对定量。 The Application of The Method- Neuron Analysis.1) Quantitative Estimation of the Amounts of Glur2 MRNAS IN Rat Forebrain And CerebellumThe Method Wased on the co- Amplification of an in in in in in in in in in in the single baskin from the turtleGETED gl2 D…更多的Na，其中在2098年从C到G的单基本交换是为了破坏野外的BSP1286I，创建了Aunique Stui位点，并用作内标的。酶从内源性靶标mRNA和DNA衍生的DNA衍生而来，由凝胶电泳衍生而来的DNA。大鼠小脑和小脑包含1.0x 10^5和1.6 x 10^4副本的glur2 mRNA/ng rna。内标被应用于来自11个神经元中大鼠的单次培养基中的单个锥体神经元中表达的AMOUR2 mRNA，GLUR2 mRNA的副本数量从100到3000不等。平均数量为1015*317（平均*SE）。较少的