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

我们以前已经表明，天然AMPA型谷氨酸受体的功能特性的分子基础，可以在单细胞水平上进行分析，通过使用膜片钳逆转录聚合酶链反应（膜片钳RT-PCR）技术。然而，先前的方法不允许定量估计单个细胞中表达的mRNA的量。本研究旨在建立一种在单细胞水平上对脑神经元GluR 2 AMPA受体亚基mRNA进行绝对定量的方法。研究结果分为以下两部分，本文首先介绍了定量方法的建立，然后介绍了单神经元分析方法的应用。1）定量测定大鼠前脑和小脑中GluR 2 mRNA的含量。扩增体外产生的转录物，其与靶向GluR 2 mRNA的差异在于单个碱基变化。 ...更多信息 NA作为内标物，其中在2098位进行了从C到G的单碱基交换，以破坏野生型的Bsp 1286 I并产生独特的StuI位点。在转化为cDNA和PCR后，扩增的DNA产物可以用适当的限制性内切酶消化，以通过凝胶电泳区分源自内源性靶mRNA的DNA和源自内标RNA的DNA。用于PCR的反义引物用13 P标记<32>，消化的PCR产物的量用BAS 2000（Fuji Film）定量。通过该方法估计，大鼠前脑和小脑含有1.0 × 10^5和1.6 × 10^4拷贝的GluR 2 mRNA/ng RNA，2）单个培养海马神经元中GluR 2 mRNA表达量的定量估计应用PCR技术，以内标法测定大鼠胚胎原代培养的单个锥体神经元中GluR 2 mRNA的表达量。在11个神经元中，GluR 2 mRNA的拷贝数从100到3000不等。平均数为1015*317（平均值 *SE）。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