Devienne Gabrielle, Le Gac Benjamin, Piquet Juliette, Cauli Bruno
UPMC Univ Paris 06, INSERM, CNRS, Neuroscience Paris Seine - Institut de Biologie Paris Seine (NPS - IBPS), Sorbonne Université.
UPMC Univ Paris 06, INSERM, CNRS, Neuroscience Paris Seine - Institut de Biologie Paris Seine (NPS - IBPS), Sorbonne Université;
J Vis Exp. 2018 Jun 20(136):57627. doi: 10.3791/57627.
The cerebral cortex is composed of numerous cell types exhibiting various morphological, physiological, and molecular features. This diversity hampers easy identification and characterization of these cell types, prerequisites to study their specific functions. This article describes the multiplex single cell reverse transcription polymerase chain reaction (RT-PCR) protocol, which allows, after patch-clamp recording in slices, to detect simultaneously the expression of tens of genes in a single cell. This simple method can be implemented with morphological characterization and is widely applicable to determine the phenotypic traits of various cell types and their particular cellular environment, such as in the vicinity of blood vessels. The principle of this protocol is to record a cell with the patch-clamp technique, to harvest and reverse transcribe its cytoplasmic content, and to detect qualitatively the expression of a predefined set of genes by multiplex PCR. It requires a careful design of PCR primers and intracellular patch-clamp solution compatible with RT-PCR. To ensure a selective and reliable transcript detection, this technique also requires appropriate controls from cytoplasm harvesting to amplification steps. Although precautions discussed here must be strictly followed, virtually any electrophysiological laboratory can use the multiplex single cell RT-PCR technique.
大脑皮层由众多具有各种形态、生理和分子特征的细胞类型组成。这种多样性阻碍了对这些细胞类型的轻松识别和表征，而这些细胞类型的识别和表征是研究其特定功能的前提条件。本文描述了多重单细胞逆转录聚合酶链反应（RT-PCR）方案，该方案允许在脑片膜片钳记录后，在单个细胞中同时检测数十个基因的表达。这种简单的方法可以与形态学表征相结合，广泛适用于确定各种细胞类型的表型特征及其特定的细胞环境，如血管附近的环境。该方案的原理是用膜片钳技术记录一个细胞，收获并逆转录其细胞质内容物，然后通过多重PCR定性检测一组预定义基因的表达。它需要精心设计与RT-PCR兼容的PCR引物和细胞内膜片钳溶液。为确保选择性和可靠的转录本检测，该技术还需要从细胞质收获到扩增步骤进行适当的对照。尽管必须严格遵循此处讨论的预防措施，但几乎任何电生理实验室都可以使用多重单细胞RT-PCR技术。
