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An optimized protocol using Steedman's wax for high-sensitivity RNA in situ hybridization in shoot apical meristems and flower buds of cucumber

文献类型： 外文期刊

作者： Wang Cui 1 ; Sun Jin-jing 2 ; Yang Xue-yong 2 ; Wan Li 2 ; Zhang Zhong-hua 1 ; Zhang Hui-min 1 ;

作者机构： 1.Qingdao Agr Univ, Coll Hort, Engn Lab Genet Improvement Hort Crops Shandong Pr, Minist Agr & Rural Affairs,Lab Qual & Safety Risk, Qingdao 266109, Peoples R China

2.Chinese Acad Agr Sci, Inst Vegetables & Flowers, Key Lab Biol & Genet Improvement Hort Crops, Minist Agr & Rural Affairs,Sino Dutch Joint Lab H, Beijing 100081, Peoples R China

关键词： cucumber; in situ hybridization; Steedman's wax; paraffin wax

期刊名称：JOURNAL OF INTEGRATIVE AGRICULTURE （ 影响因子：4.8； 五年影响因子：4.8 ）

ISSN： 2095-3119

年卷期： 2023 年 22 卷 2 期

页码：

收录情况： SCI

摘要： In situ mRNA 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 the CLAVATA3 (CLV3) and WUSCHEL (WUS) genes in shoot apical meristems and floral meristems of Cucumis sativus (cucumber) and Arabidopsis 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.

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