Stowers Institute for Medical Research, Kansas City, MO 64110, USA.
Department of Anatomy and Cell Biology, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, KS 66160, USA.
Cell Rep Methods. 2022 Apr 18;2(4):100201. doi: 10.1016/j.crmeth.2022.100201. eCollection 2022 Apr 25.
We describe a cost-effective, highly sensitive, and quantitative method for detection of RNA molecules in tissue sections. This method, dubbed Yn-situ, standing for Y-branched probe 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.
我们描述了一种经济高效、高度敏感且定量的方法，用于检测组织切片中的 RNA 分子。这种方法被称为 Yn-situ，代表 Y 型探针杂交，使用具有多个起始位点的单链 DNA 预扩增子来触发杂交链式反应 (HCR) 以检测多核苷酸。通过对该方法的性能进行表征，我们表明，Yn-situ 方法与改进的固定步骤相结合，足够灵敏，允许使用针对短核苷酸序列的较少探针来检测 RNA 分子，而不是现有方法。一组五个探针可以产生比 HCR 和 RNAscope 通常需要的 20 个探针组更小的斑点和更高的信噪比的定量结果。我们表明，高灵敏度和宽动态范围允许定量检测嗅觉感觉神经元中不同水平表达的基因。我们描述了该方法的关键步骤，以使其能够被各个实验室广泛使用。
