Esumi Shigeyuki, Kaneko Ryosuke, Kawamura Yoshimi, Yagi Takeshi
KOKORO-Biology Group, Laboratories for Integrated Biology, Graduate School of Frontier Biosciences, Osaka University 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan.
Nat Protoc. 2006;1(4):2143-51. doi: 10.1038/nprot.2006.343.
This protocol details a method for analyzing the expression of multiple genes from a single Purkinje neuron, including the determination of whether the gene expression is monoallelic or biallelic. The protocol describes how to extract a single, living Purkinje cell for reverse transcription, divide the cDNAs into three equal samples and subject those to triplicate amplification of multiple targets by two rounds of PCR (first a multiplex PCR then a gene-specific nested PCR) and finally discriminate the allelic expression of the transcript by direct sequencing of the PCR products. In optimal conditions, this method permits the analysis of the expression of 18 genes in a single Purkinje cell. This protocol can be completed in 5-6 d.
本方案详细介绍了一种分析单个浦肯野神经元中多个基因表达的方法，包括确定基因表达是单等位基因还是双等位基因。该方案描述了如何提取单个活的浦肯野细胞用于逆转录，将cDNA分成三个相等的样本，并通过两轮PCR（首先是多重PCR，然后是基因特异性巢式PCR）对这些样本进行多个靶标的三重扩增，最后通过对PCR产物进行直接测序来鉴别转录本的等位基因表达。在最佳条件下，该方法允许分析单个浦肯野细胞中18个基因的表达。本方案可在5-6天内完成。
