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Establishment and optimization of mRNA in situ hybridization system in turnip (Brassica rapa var. rapa)
Li,Cheng1,2,3; Hu,Simin1,2,3; Lei,Qidong4; Wang,Chongde5; Yang,Yunqiang1,2; Yang,Yongping1,2; Sun,Xudong1,2
Corresponding AuthorYang,Yongping(yangyp@mail.kib.ac.cn) ; Sun,Xudong(sunxudong@mail.kib.ac.cn)
2019-10-16
Source PublicationPlant Methods
ISSN1746-4811
Volume15Issue:1Pages:10
AbstractAbstractBackgroundIn situ hybridization (ISH) is a general molecular biological technique used to determine the spatiotemporal expression of genes in many species. In the past few years, numerous ISH protocols have been established in many species. Turnip (Brassica rapa var. rapa) is an important crop in the world, especially in the Plateau area of China, and is a traditional Tibetan medicine. However, ISH protocol in turnip has not been established.ResultsWe explored and established an optimal workflow for mRNA ISH system for turnip which has been evaluated using BrrCLV3 and BrrWUSa. The optimal methods include: (1) fixation method, (2) protease K pretreatment time, (3) probe length and concentration, (4) washing temperature. We also provide advice on weakening background and improving the efficiency of RNA transcription in vitro. The expression of the BrrCLV3 gene in turnip was detected by the optimized system, and the applicability of the system was confirmed by using BrrWUSa.ConclusionsIn this study, we established and optimized the mRNA ISH system for turnip. We explored and found that (1) FAA fixative was the optimized fixation method, (2) 30?min was the optimized protease K pretreatment time, (3) 100?bp, 100?ng/ml probe had good hybridization signal, (4) the optimized washing temperature was 52?°C. It provides a powerful method to locate mRNA in the tissue, which can study the expression and function of turnip’s genes. As such, it has considerable advantages in terms of time and cost.
KeywordmRNA in situ hybridization Brassica rapa var. rapa Gene expression BrrCLV3 BrrWUSa
DOI10.1186/s13007-019-0499-4
Indexed BySCI ; SCI
Language英语
WOS Research AreaBiochemistry & Molecular Biology ; Plant Sciences
WOS SubjectBiochemical Research Methods ; Plant Sciences
WOS IDBMC:10.1186/s13007-019-0499-4
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Document Type期刊论文
Identifierhttp://ir.kib.ac.cn/handle/151853/68403
Collection中国西南野生生物种质资源库
Corresponding AuthorYang,Yongping; Sun,Xudong
Affiliation1.
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Recommended Citation
GB/T 7714
Li,Cheng,Hu,Simin,Lei,Qidong,et al. Establishment and optimization of mRNA in situ hybridization system in turnip (Brassica rapa var. rapa)[J]. Plant Methods,2019,15(1):10.
APA Li,Cheng.,Hu,Simin.,Lei,Qidong.,Wang,Chongde.,Yang,Yunqiang.,...&Sun,Xudong.(2019).Establishment and optimization of mRNA in situ hybridization system in turnip (Brassica rapa var. rapa).Plant Methods,15(1),10.
MLA Li,Cheng,et al."Establishment and optimization of mRNA in situ hybridization system in turnip (Brassica rapa var. rapa)".Plant Methods 15.1(2019):10.
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