Establishment and optimization of mRNA in situ hybridization system in turnip (Brassica rapa var. rapa)
Abstract Background In 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...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2019-10-01
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| Series: | Plant Methods |
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| Online Access: | http://link.springer.com/article/10.1186/s13007-019-0499-4 |
| _version_ | 1818883216366370816 |
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| author | Cheng Li Simin Hu Qidong Lei Chongde Wang Yunqiang Yang Yongping Yang Xudong Sun |
| author_facet | Cheng Li Simin Hu Qidong Lei Chongde Wang Yunqiang Yang Yongping Yang Xudong Sun |
| author_sort | Cheng Li |
| collection | DOAJ |
| description | Abstract Background In 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. Results We 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. Conclusions In 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. |
| first_indexed | 2024-12-19T15:30:07Z |
| format | Article |
| id | doaj.art-8c32feda68994784a7c9e85040597d74 |
| institution | Directory Open Access Journal |
| issn | 1746-4811 |
| language | English |
| last_indexed | 2024-12-19T15:30:07Z |
| publishDate | 2019-10-01 |
| publisher | BMC |
| record_format | Article |
| series | Plant Methods |
| spelling | doaj.art-8c32feda68994784a7c9e85040597d742022-12-21T20:15:46ZengBMCPlant Methods1746-48112019-10-0115111010.1186/s13007-019-0499-4Establishment and optimization of mRNA in situ hybridization system in turnip (Brassica rapa var. rapa)Cheng Li0Simin Hu1Qidong Lei2Chongde Wang3Yunqiang Yang4Yongping Yang5Xudong Sun6Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of SciencesKey Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of SciencesFaculty of Life Science and Technology, Kunming University of Science and TechnologyCollege of Plant Protection, Yunnan Agriculture UniversityKey Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of SciencesKey Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of SciencesKey Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of SciencesAbstract Background In 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. Results We 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. Conclusions In 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.http://link.springer.com/article/10.1186/s13007-019-0499-4mRNA in situ hybridizationBrassica rapa var. rapaGene expressionBrrCLV3BrrWUSa |
| spellingShingle | Cheng Li Simin Hu Qidong Lei Chongde Wang Yunqiang Yang Yongping Yang Xudong Sun Establishment and optimization of mRNA in situ hybridization system in turnip (Brassica rapa var. rapa) Plant Methods mRNA in situ hybridization Brassica rapa var. rapa Gene expression BrrCLV3 BrrWUSa |
| title | Establishment and optimization of mRNA in situ hybridization system in turnip (Brassica rapa var. rapa) |
| title_full | Establishment and optimization of mRNA in situ hybridization system in turnip (Brassica rapa var. rapa) |
| title_fullStr | Establishment and optimization of mRNA in situ hybridization system in turnip (Brassica rapa var. rapa) |
| title_full_unstemmed | Establishment and optimization of mRNA in situ hybridization system in turnip (Brassica rapa var. rapa) |
| title_short | Establishment and optimization of mRNA in situ hybridization system in turnip (Brassica rapa var. rapa) |
| title_sort | establishment and optimization of mrna in situ hybridization system in turnip brassica rapa var rapa |
| topic | mRNA in situ hybridization Brassica rapa var. rapa Gene expression BrrCLV3 BrrWUSa |
| url | http://link.springer.com/article/10.1186/s13007-019-0499-4 |
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