Mistry Ragini M, Singh Pankaj K, Mancini Maureen G, Stossi Fabio, Mancini Michael A
GCC Center for Advanced Microscopy and Image Informatics; Department of Molecular and Cellular Biology, Baylor College of Medicine.
GCC Center for Advanced Microscopy and Image Informatics; Center for Translational Cancer Research, Institute of Biosciences and Technology, Texas A&M University.
J Vis Exp. 2020 Sep 20(163). doi: 10.3791/61680.
Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk population methods (Northern blot, PCR, and RNAseq) that measure mRNA levels lack the ability to provide information on cell-to-cell variation in responses. Precise single cell and allelic visualization and quantification is possible via single molecule RNA fluorescence in situ hybridization (smFISH). RNA-FISH is performed by hybridizing target RNAs with labeled oligonucleotide probes. These can be imaged in medium/high throughput modalities, and, through image analysis pipelines, provide quantitative data on both mature and nascent RNAs, all at the single cell level. The fixation, permeabilization, hybridization and imaging steps have been optimized in the lab over many years using the model system described herein, which results in successful and robust single cell analysis of smFISH labeling. The main goal with sample preparation and processing is to produce high quality images characterized by a high signal-to-noise ratio to reduce false positives and provide data that are more accurate. Here, we present a protocol describing the pipeline from sample preparation to data analysis in conjunction with suggestions and optimization steps to tailor to specific samples.
基因转录是细胞生物学中的一个基本过程，它使细胞能够解读并响应内部和外部信号。传统的用于测量mRNA水平的大量细胞群体方法（Northern印迹法、聚合酶链式反应和RNA测序）无法提供细胞间反应差异的信息。通过单分子RNA荧光原位杂交（smFISH）可以实现精确的单细胞和等位基因可视化及定量分析。RNA荧光原位杂交是通过将靶RNA与标记的寡核苷酸探针杂交来进行的。这些探针可以通过中/高通量方式成像，并通过图像分析流程在单细胞水平上提供有关成熟RNA和新生RNA的定量数据。多年来，我们实验室使用本文所述的模型系统对固定、通透、杂交和成像步骤进行了优化，从而实现了对smFISH标记的成功且稳健的单细胞分析。样品制备和处理的主要目标是生成具有高信噪比的高质量图像，以减少假阳性并提供更准确的数据。在此，我们提供了一个方案，描述了从样品制备到数据分析的流程，并结合了针对特定样品的建议和优化步骤。
