1] Faculty of Sciences, Institute of Molecular Life Sciences, University of Zurich, Zurich, Switzerland. [2] Systems Biology PhD program, Life Science Zurich Graduate School, ETH Zurich and University of Zurich, Zurich, Switzerland. [3].
Nat Methods. 2013 Nov;10(11):1127-33. doi: 10.1038/nmeth.2657. Epub 2013 Oct 6.
Fluorescence in situ hybridization (FISH) is widely used to obtain information about transcript copy number and subcellular localization in single cells. However, current approaches do not readily scale to the analysis of whole transcriptomes. Here we show that branched DNA technology combined with automated liquid handling, high-content imaging and quantitative image analysis allows highly reproducible quantification of transcript abundance in thousands of single cells at single-molecule resolution. In addition, it allows extraction of a multivariate feature set quantifying subcellular patterning and spatial properties of transcripts and their cell-to-cell variability. This has multiple implications for the functional interpretation of cell-to-cell variability in gene expression and enables the unbiased identification of functionally relevant in situ signatures of the transcriptome without the need for perturbations. Because this method can be incorporated in a wide variety of high-throughput image-based approaches, we expect it to be broadly applicable.
荧光原位杂交（FISH）被广泛用于获取单细胞中转录物拷贝数和亚细胞定位的信息。然而，当前的方法不容易扩展到整个转录组的分析。在这里，我们展示了分支 DNA 技术与自动化液体处理、高内涵成像和定量图像分析相结合，可以以单分子分辨率对数千个单细胞中的转录物丰度进行高度可重复的定量。此外，它还可以提取一个多变量特征集，定量转录物的亚细胞模式和空间特性及其细胞间变异性。这对功能解释细胞间基因表达变异性具有多种意义，并能够在不需要干扰的情况下，无偏地识别转录组中与功能相关的原位特征。由于该方法可以整合到各种高通量基于图像的方法中，因此我们预计它具有广泛的适用性。
