Huang Kun, Batish Mona, Teng Chong, Harkess Alex, Meyers Blake C, Caplan Jeffrey L
Department of Plant and Soil Sciences, University of Delaware, Newark, DE, USA.
Delaware Biotechnology Institute, University of Delaware, Newark, DE, USA.
Methods Mol Biol. 2020;2166:23-33. doi: 10.1007/978-1-0716-0712-1_2.
Single-molecule FISH (smFISH) has been widely used in animal tissue to localize and quantify RNAs with high specificity. This protocol describes an smFISH method optimized for highly autofluorescent plant tissue. It provides details on fixation buffers and protocols to protect the integrity of plant samples. We also provide smFISH hybridization conditions to detect plant RNA with ~50 fluorescently labeled DNA oligonucleotides. In addition, this protocol provides instructions on linear spectral unmixing of smFISH signal from background autofluorescence by confocal microscopy and a method to quantify the smFISH spots that reflect the copy number of target RNA.
单分子荧光原位杂交（smFISH）已广泛应用于动物组织，以高特异性地定位和定量RNA。本方案描述了一种针对高自发荧光植物组织优化的smFISH方法。它提供了固定缓冲液和保护植物样品完整性的方案的详细信息。我们还提供了使用约50个荧光标记的DNA寡核苷酸检测植物RNA的smFISH杂交条件。此外，本方案提供了通过共聚焦显微镜对smFISH信号与背景自发荧光进行线性光谱解混的说明，以及一种量化反映靶RNA拷贝数的smFISH斑点的方法。
