An optimized protocol using Steedman's wax for high-sensitivity RNA in situ hybridization in shoot apical meristems and flower buds of cucumber
An optimized protocol using Steedman's wax for high-sensitivity RNA in situ hybridization in shoot apical meristems and flower buds of cucumber
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DOI：
10.1016/j.jia.2022.08.038
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发表时间：
2023-01-26
影响因子：
4.8
通讯作者：
Zhang Hui-min，yan
中科院分区：
文献类型：
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作者：
Wang Cui，Cui;Sun Jin-jing，Jin-jing;Zhang Hui-min，yan
原位RNA杂交（ISH）是研究黄瓜茎尖分生组织和花芽基因时空表达的有力工具。最常见的ISH方案使用石蜡;然而，将组织包埋在石蜡中可能需要很长时间，并且可能导致RNA降解和信号降低。在这里，我们开发了一种优化的方案来简化过程并提高RNA灵敏度。我们结合包埋组织在低熔点Steedman的蜡处理组织切片在溶液中，在整个安装ISH方法的优化协议。利用优化的表达方案，我们检测了CLAVATA 3（CLV 3）和WUSCHEL（WUS）基因在黄瓜（Cucumis sativus）和拟南芥（Arabidopsis thaliana）茎尖分生组织和花分生组织中的表达模式。与石蜡包埋的ISH方法相比，优化后的ISH方法缩短了4-5天的实验时间。此外，优化的方案实现了高的信号灵敏度。优化后的反应体系在黄瓜和拟南芥上均获得了成功，表明该反应体系对大多数植物具有普遍适用性。
In situmRNA hybridization (ISH) is a powerful tool for examining the spatiotemporal expression of genes in shoot apical meristems and flower buds of cucumber. The most common ISH protocol uses paraffin wax; however, embedding tissue in paraffin wax can take a long time and might result in RNA degradation and decreased signals. Here, we developed an optimized protocol to simplify the process and improve RNA sensitivity. We combined embedding tissue in low melting-point Steedman's wax with processing tissue sections in solution, as in the whole-mount ISH method in the optimized protocol. Using the optimized protocol, we examined the expression patterns of theCLAVATA3(CLV3) andWUSCHEL(WUS) genes in shoot apical meristems and floral meristems ofCucumis sativus(cucumber) andArabidopsis thaliana(Arabidopsis). The optimized protocol saved 4–5 days of experimental period compared with the standard ISH protocol using paraffin wax. Moreover, the optimized protocol achieved high signal sensitivity. The optimized protocol was successful for both cucumber and Arabidopsis, which indicates it might have general applicability to most plants.