Department of Biochemistry, University of Oxford, Oxford, UK.
School of Molecular Biosciences, College of Medical, Veterinary & Life Sciences, University of Glasgow, Glasgow, UK.
Fly (Austin). 2024 Dec;18(1):2409968. doi: 10.1080/19336934.2024.2409968. Epub 2024 Oct 1.
hybridization techniques are powerful methods for exploring gene expression in a wide range of biological contexts, providing spatial information that is most often lost in traditional biochemical techniques. However, many hybridization methods are costly and time-inefficient, particularly for screening-based projects that follow on from single-cell RNA sequencing data, which rely on of tens of custom-synthetized probes against each specific RNA of interest. Here we provide an optimized pipeline for Hybridization Chain Reaction (HCR)-based RNA visualization, including an open-source code for optimized probe design. Our method achieves high specificity and sensitivity with the option of multiplexing using only five pairs of probes, which greatly lowers the cost and time of the experiment. These features of our HCR protocol are particularly useful and convenient for projects involving screening several genes at medium throughput, especially as the method include an amplification step, which makes the signal readily visible at low magnification imaging.
杂交技术是探索广泛生物背景下基因表达的强大方法，提供了传统生化技术中经常丢失的空间信息。然而，许多杂交方法成本高且效率低，特别是对于基于筛选的项目，这些项目是在单细胞 RNA 测序数据之后进行的，它们依赖于针对每个特定感兴趣 RNA 合成的数十个定制探针。在这里，我们提供了一种基于杂交链反应 (HCR) 的 RNA 可视化的优化流程，包括用于优化探针设计的开源代码。我们的方法实现了高特异性和灵敏度，并且可以使用仅五对探针进行多重化，这大大降低了实验的成本和时间。我们的 HCR 方案的这些特点对于涉及以中等通量筛选几个基因的项目特别有用和方便，特别是因为该方法包括一个扩增步骤，使得信号在低倍放大成像时很容易看到。
