Detecting Spaciotemporal Transcript Accumulation in Maize by RNA In Situ Hybridization
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Author:
Date:
2021-02-20
[Abstract] RNA in situ hybridization is a method for visualizing spatiotemporal transcript accumulation in cells and tissues. The method provides clear resolution, is highly sensitive and specific, and can uncover gradients of transcript accumulation within a histologically-intact tissue, which is not possible currently with other methods for transcript detection. RNA in situ hybridization, however, is not a quantitative approach for gene expression. Protocols for RNA in situ hybridization have numerous steps that can span several days of work, complicating troubleshooting procedures. Here, we build on previously published RNA in situ hybridization protocols optimized for paraffin-embedded and sectioned maize tissue (Jackson, 1991; Long et al., 1996 ; ...
[摘要] [摘要] RNA原位杂交是一种可视化时空转录本在细胞和组织中积累的方法。该方法提供清晰的分辨率,高度灵敏和特异,并且可以揭示组织完整的组织内转录物积累的梯度,这是当前其他方法无法检测到转录物的方法。然而,RNA原位杂交不是用于基因表达的定量方法。RNA原位杂交的协议有许多步骤,可能需要花费数天的时间,从而使故障排除过程变得更加复杂。在这里,我们基于先前发布的RNA原位构建 杂交方案针对石蜡包埋和切片的玉米组织进行了优化(Jackson,1991; Long等,1996;Javelle等,2011),它提供了优化转录本检测的其他措施。
图形摘要:
RNA原位杂交的工作流程
[背景技术]在时间和空间差异基因表达允许具有相同的遗传物质的细胞呈现不同的身份。确实,基因表达的这种变化通常负责驱动整个生物体中模式或形态的进化变化(Carroll,2005)。因此,通过观察组织内天然背景下的基因表达,可以增强对发育过程的见识。诸如RT-qPCR和RNA- ...
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In vitro assessment of ivermectin resistance and gene expression profiles of P-glycoprotein genes from Haemonchus contortus (L3)
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Author:
Date:
2020-12-20
[Abstract] The aim of the present protocol is to improve the methodology to identify resistance to ivermectin (IVM), one of the main anthelmintic drugs against parasitic nematodes of ruminants, using the blood-feeding nematode Haemonchus contortus as biological model. The infective larvae (L3) of H. contortus are frequently selected to perform in vitro and molecular assays to analyze problems with drug resistance. This protocol describes the procedures to conserve the integrity and quality of H. contortus larvae in different experimental assays. Two nematode isolates, resistant and susceptible to IVM, are compared to estimate the lethal effect using IVM at 1.43, 2.85, 5.71 and 11.42 mM dilutions in the non-ionic detergent Triton X-100 to conserve the ...
[摘要] [摘要]本协议的目的是使用血液喂养的线虫Haemonchus contortus作为生物学模型,改进鉴定对伊维菌素(IVM)的抗药性的方法,伊维菌素是针对反刍动物寄生线虫的主要驱虫药之一。的感染性幼虫(L 3 )的捻转血矛线虫,经常选择为PE rform体外和分子测定来分析与药物抗性的问题。该协议描述了保存捻转血吸虫完整性和质量的程序幼虫在不同的实验分析中。比较了两种对IVM具有抗药性和易感性的线虫分离物,以在非离子型清洁剂Triton X-100中以1.43、2.85、5.71和11.42 mM的IVM稀释度评估IVM的致死作用,以保留实验期间对照幼虫的活性。 (从24小时到72小时)。在过去的几十年中,使用IVM和其他驱虫药诊断的重要性要求确认易感菌株以评估最佳控制策略。中号矿石诊断的灵敏的方法是亲通过PCR技术和其他分子工具,如测序vided。在该方案中,使用10个主要的P-糖蛋白基因(1、2、3、4、9、10 ,11、12、14和16),使用逆转录和实时PCR(RT-qPCR),在与易受IVM侵害的线虫分离株相比,确认了捻转嗜血杆菌对IVM的抵抗力。
[背景] d ...
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RNA Immunoprecipitation (RIP) Sequencing of Pri-miRNAs Associated with the Dicing Complex in Arabidopsis
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Author:
Date:
2018-07-05
[Abstract] RNA immunoprecipitation (RIP) is an antibody-based technique used to map in vivo RNA-protein interactions. DBR1, an RNA debranching enzyme, is responsible for the debranching of lariat RNA, for the degradation and turnover of lariat RNAs. It is well known that primary miRNA (Pri-miRNA) is recognized and further processed into mature miRNA by the Dicing complex mainly composed of DCL1 and HYL1. Due to the low abundance of pri-miRNAs, RIP followed qRT-PCR has been widely used to evaluate the binding efficiency of the Dicing complex with pri-miRNAs in previous studies. Therefore, the genome-wide evaluation of the Dicing complex with pri-miRNAs is lacking. With the improvement of high-throughput sequencing technologies, we successfully used RIP-seq to compare the binding efficiency ...
[摘要] RNA免疫沉淀(RIP)是一种基于抗体的技术,用于绘制体内 RNA-蛋白质相互作用。 DBR1是一种RNA脱支酶,负责套索RNA的脱支,用于套索RNA的降解和转换。众所周知,主要miRNA(Pri-miRNA)被主要由DCL1和HYL1组成的切割复合物识别并进一步加工成成熟miRNA。由于pri-miRNA的丰度较低,RIP随后的qRT-PCR已被广泛用于评估切割复合物与pri-miRNA在先前研究中的结合效率。因此,缺乏对具有pri-miRNA的切割复合物的全基因组评估。随着高通量测序技术的改进,我们成功地使用RIP-seq比较了Dicing复合物与野生型和 dbr1-2 突变体之间的pri-miRNA的结合效率。 。在该方案中,我们提供了在两种不同基因型之间的HYL1-YFP和DCL1-YFP转基因植物中使用GFP捕获珠的RIP-seq的详细描述。该方法可用于评估pri-miRNA与拟南芥中的切割复合物的结合,并且它可以应用于植物中的其他RNA结合蛋白。
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