{"title":"利用原位RT-PCR技术检测油菜保护细胞特异性基因表达的优化方案","authors":"Yingying Song, Xinlei Guo, Jian Wu, Jianli Liang, Runmao Lin, Zifu Yan, Xiaowu Wang","doi":"10.21769/BioProtoc.4810","DOIUrl":null,"url":null,"abstract":"<p><p>Since the genetic transformation of Chinese cabbage (<i>Brassica rapa</i>) has not been well developed, in situ RT-PCR is a valuable option for detecting guard cell-specific genes. We reported an optimized protocol of in situ RT-PCR by using a FAMA homologous gene Bra001929 in Brassica rapa. FAMA in Arabidopsis has been verified to be especially expressed in guard cells. We designed specific RT-PCR primers and optimized the protocol in terms of the (a) reverse transcription time, (b) blocking time, (c) antigen-antibody incubation time, and (d) washing temperature. Our approach provides a sensitive and effective in situ RT-PCR method that can detect low-abundance transcripts in cells by elevating their levels by RT-PCR in the guard cells in Brassica rapa.</p>","PeriodicalId":8938,"journal":{"name":"Bio-protocol","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"2023-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/dc/f0/BioProtoc-13-17-4810.PMC10501917.pdf","citationCount":"0","resultStr":"{\"title\":\"An Optimized Protocol for Detecting Guard Cell-specific Gene Expression by in situ RT-PCR in <i>Brassica rapa</i>.\",\"authors\":\"Yingying Song, Xinlei Guo, Jian Wu, Jianli Liang, Runmao Lin, Zifu Yan, Xiaowu Wang\",\"doi\":\"10.21769/BioProtoc.4810\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>Since the genetic transformation of Chinese cabbage (<i>Brassica rapa</i>) has not been well developed, in situ RT-PCR is a valuable option for detecting guard cell-specific genes. We reported an optimized protocol of in situ RT-PCR by using a FAMA homologous gene Bra001929 in Brassica rapa. FAMA in Arabidopsis has been verified to be especially expressed in guard cells. We designed specific RT-PCR primers and optimized the protocol in terms of the (a) reverse transcription time, (b) blocking time, (c) antigen-antibody incubation time, and (d) washing temperature. Our approach provides a sensitive and effective in situ RT-PCR method that can detect low-abundance transcripts in cells by elevating their levels by RT-PCR in the guard cells in Brassica rapa.</p>\",\"PeriodicalId\":8938,\"journal\":{\"name\":\"Bio-protocol\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2023-09-05\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/dc/f0/BioProtoc-13-17-4810.PMC10501917.pdf\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Bio-protocol\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.21769/BioProtoc.4810\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"\",\"JCRName\":\"\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Bio-protocol","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.21769/BioProtoc.4810","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
An Optimized Protocol for Detecting Guard Cell-specific Gene Expression by in situ RT-PCR in Brassica rapa.
Since the genetic transformation of Chinese cabbage (Brassica rapa) has not been well developed, in situ RT-PCR is a valuable option for detecting guard cell-specific genes. We reported an optimized protocol of in situ RT-PCR by using a FAMA homologous gene Bra001929 in Brassica rapa. FAMA in Arabidopsis has been verified to be especially expressed in guard cells. We designed specific RT-PCR primers and optimized the protocol in terms of the (a) reverse transcription time, (b) blocking time, (c) antigen-antibody incubation time, and (d) washing temperature. Our approach provides a sensitive and effective in situ RT-PCR method that can detect low-abundance transcripts in cells by elevating their levels by RT-PCR in the guard cells in Brassica rapa.