以单转录本分辨率对活体系统中的实时基因表达进行成像：单个mRNA转录本的图像分析
Imaging real-time gene expression in living systems with single-transcript resolution: image analysis of single mRNA transcripts.
作者信息
Wells Amber L, Condeelis John S, Singer Robert H, Zenklusen Daniel
机构信息
Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
出版信息
CSH Protoc. 2007 Nov 1;2007:pdb.prot4871. doi: 10.1101/pdb.prot4871.
INTRODUCTIONThis protocol describes a method for establishing a green fluorescent protein (GFP) calibration curve using dilutions of recombinant GFP and blue fluorescent beads. The total fluorescence intensity (TFI) per mRNA molecule is first calculated by imaging serial dilutions of purified enhanced GFP (eGFP) to determine the TFI within a specific volume. A calibration curve of fluorescence intensity in a given voxel per molecule of GFP is then used to determine the number of GFP molecules in the sample of formaldehyde fixed cells to be imaged. This is followed by a method for detection of single molecules in formaldehyde-fixed and live cells. These cells have been cotransfected with mRNA reporter and MCP-xFP plasmids, where MCP-xFP refers to a fluorescent protein fused to the MS2 capsid protein. It is important to collect micrographs and establish the calibration curve on the same day that the cells are imaged, using the same equipment configuration, camera settings, and image acquisition parameters.
引言
本方案描述了一种使用重组绿色荧光蛋白（GFP）稀释液和蓝色荧光珠建立绿色荧光蛋白校准曲线的方法。首先通过对纯化的增强型绿色荧光蛋白（eGFP）系列稀释液进行成像，计算每个mRNA分子的总荧光强度（TFI），以确定特定体积内的TFI。然后使用每分子GFP在给定体素中的荧光强度校准曲线，来确定待成像的甲醛固定细胞样品中的GFP分子数量。接下来是一种检测甲醛固定细胞和活细胞中单个分子的方法。这些细胞已与mRNA报告基因和MCP-xFP质粒共转染，其中MCP-xFP是指与MS2衣壳蛋白融合的荧光蛋白。在细胞成像的同一天，使用相同的设备配置、相机设置和图像采集参数收集显微照片并建立校准曲线非常重要。
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