Robust and sensitive in situ RNA detection using Yn-situ.
Robust and sensitive in situ RNA detection using Yn-situ.
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DOI：
10.1016/j.crmeth.2022.100201
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发表时间：
2022-04-25
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我们描述了一种具有成本效益的，高灵敏度的，和定量的方法在组织切片中的RNA分子的原位检测。这种方法被称为Yn-situ，代表Y分支探针原位杂交，使用具有多个起始位点的单链DNA前置放大器，触发杂交链反应（HCR）来检测多核苷酸。通过表征这种方法的性能，我们表明，与改进的固定步骤相结合的Yn原位方法，是足够灵敏的，允许使用更少的探针靶向短核苷酸序列比现有的方法检测RNA分子。与HCR和RNAscope通常需要的20个探针组相比，一组5个探针可以产生具有更小斑点和更高信噪比的定量结果。我们表明，高灵敏度和宽的动态范围允许量化的基因表达在不同水平的嗅觉感觉神经元。我们描述了这种方法的关键步骤，使个别实验室的广泛应用。前置放大器提高了灵敏度和信噪比，减少了斑点大小Yn-situ是稳健的，具有成本效益，易于使用实现蛋白质和RNA的同时检测原位杂交广泛用于检测细胞RNA。使用短寡核苷酸探针的现代方法已经消除了长RNA探针的使用，但是许多短探针的需求限制了长转录物的应用，并且当使用专有试剂时导致高成本。在目前的研究中，我们的目的是提高鲁棒性，灵敏度和易用性，并降低目前的单分子荧光原位杂交方法的成本。Wu等人开发Yn-situ，一种易于使用且具有成本效益的原位RNA检测方法。作者介绍了一个固定步骤，以提高原位杂交方法的灵敏度和鲁棒性。
We describe a cost-effective, highly sensitive, and quantitative method for in situ detection of RNA molecules in tissue sections. This method, dubbed Yn-situ, standing for Y-branched probe in situ hybridization, uses a single-strand DNA preamplifier with multiple initiation sites that trigger a hybridization chain reaction (HCR) to detect polynucleotides. By characterizing the performance of this method, we show that the Yn-situ method, in conjunction with an improved fixation step, is sensitive enough to allow detection of RNA molecules using fewer probes targeting short nucleotide sequences than existing methods. A set of five probes can produce quantitative results with smaller puncta and higher signal-to-noise ratio than the 20-probe sets commonly required for HCR and RNAscope. We show that the high sensitivity and wide dynamic range allow quantification of genes expressed at different levels in the olfactory sensory neurons. We describe key steps of this method to enable broad utility by individual laboratories. Introduction of preamplifier improves detection of cellular RNA Preamplifier improves sensitivity and signal-to-noise ratio with reduced puncta size Yn-situ is robust, cost-effective, and easy to use Simultaneous detection of protein and RNA is achieved In situ hybridization is widely used for detection of cellular RNA. Modern methods using short oligonucleotide probes have eliminated the use of long RNA probes, but the requirement of many short probes has limited the application to long transcripts and incur high cost when proprietary reagents are used. In the current study, we aimed to improve the robustness, sensitivity, and ease of use and to reduce the cost of current single-molecule fluorescent in situ hybridization methods. Wu et al. develop Yn-situ, an in situ RNA detection method that is easy to use and cost effective. The authors introduce a preamplifier and a fixation step to improve the sensitivity and robustness of in situ hybridization methods.