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

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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