In situ analysis of biomarkers such as DNA, RNA and proteins are important for research and diagnostic purposes. At the RNA level, plant gene expression studies rely on qPCR, RNAseq and probe-based in situ hybridization (ISH). However, for ISH experiments poor stability of RNA and RNA based probes commonly results in poor detection or poor reproducibility. Recently, the development and availability of the RNAscope RNA-ISH method addressed these problems by novel signal amplification and background suppression. This method is capable of simultaneous detection of multiple target RNAs down to the single molecule level in individual cells, allowing researchers to study spatio-temporal patterning of gene expression. However, this method has not been optimized thus poorly utilized for plant specific gene expression studies which would allow for fluorescent multiplex detection. Here we provide a step-by-step method for sample collection and pretreatment optimization to perform the RNAscope assay in the leaf tissues of model monocot plant barley. We have shown the spatial distribution pattern of HvGAPDH and the low expressed disease resistance gene Rpg1 in leaf tissue sections of barley and discuss precautions that should be followed during image analysis. We have shown the ubiquitous HvGAPH and predominantly stomatal guard cell associated subsidiary cell expressed Rpg1 expression pattern in barley leaf sections and described the improve RNAscope methodology suitable for plant tissues using confocal laser microscope. By addressing the problems in the sample collection and incorporating additional sample backing steps we have significantly reduced the section detachment and experiment failure problems. Further, by reducing the time of protease treatment, we minimized the sample disintegration due to over digestion of barley tissues. RNAscope multiplex fluorescent RNA-ISH detection is well described and adapted for animal tissue samples, however due to morphological and structural differences in the plant tissues the standard protocol is deficient and required optimization. Utilizing barley specific HvGAPDH and Rpg1 RNA probes we report an optimized method which can be used for RNAscope detection to determine the spatial expression and semi-quantification of target RNAs. This optimized method will be immensely useful in other plant species such as the widely utilized Arabidopsis.

中文翻译：

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通过改良的 RNAscope 荧光原位杂交可视化植物中的空间基因表达。

DNA、RNA和蛋白质等生物标志物的原位分析对于研究和诊断目的很重要。在 RNA 水平上，植物基因表达研究依赖于 qPCR、RNAseq 和基于探针的原位杂交 (ISH)。然而，对于 ISH 实验，RNA 和基于 RNA 的探针的稳定性差通常会导致检测不佳或重现性差。最近，RNAscope RNA-ISH 方法的开发和可用性通过新的信号放大和背景抑制解决了这些问题。该方法能够同时检测单个细胞中低至单个分子水平的多个靶 RNA，使研究人员能够研究基因表达的时空模式。然而，该方法尚未优化，因此很难用于允许荧光多重检测的植物特异性基因表达研究。在这里，我们提供了一种分步方法，用于样品收集和预处理优化，以在模型单子叶植物大麦的叶组织中进行 RNAscope 测定。我们展示了大麦叶片组织切片中 HvGAPDH 和低表达抗病基因 Rpg1 的空间分布模式，并讨论了在图像分析过程中应注意的事项。我们已经展示了在大麦叶切片中普遍存在的 HvGAPH 和主要与气孔保卫细胞相关的辅助细胞表达 Rpg1 表达模式，并使用共聚焦激光显微镜描述了适用于植物组织的改进 RNAscope 方法。通过解决样品收集中的问题并结合额外的样品支持步骤，我们显着减少了切片分离和实验失败问题。此外，通过减少蛋白酶处理的时间，我们最大限度地减少了由于大麦组织过度消化而导致的样品崩解。RNAscope 多重荧光 RNA-ISH 检测得到了很好的描述并适用于动物组织样本，但是由于植物组织的形态和结构差异，标准协议存在缺陷，需要进行优化。利用大麦特异性 HvGAPDH 和 Rpg1 RNA 探针，我们报告了一种优化的方法，可用于 RNAscope 检测以确定目标 RNA 的空间表达和半定量。