| Total RNA Extraction from Grape Berry Skin for Quantitative Reverse Transcription PCR and Microarray Analysis
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Author:
Date:
2016-04-05
[Abstract] Extraction of high quality RNA is an essential step for quantitative reverse transcription PCR (qRT-PCR) and microarray analysis. However, it is not easy to extract high quality RNA from fruit materials, which contain high amounts of polysaccharides, lipids and secondary metabolites. Wan and Wilkins (1994) had developed ‘Hot Borate Method’ to isolate high quality RNA. Here, we describe a modified protocol of the ‘Hot Borate Method’ to isolate high quality RNA from grape berry skin for qRT-PCR and microarray analysis (Suzuki et al., 2015a; Suzuki et al., 2015b).
[摘要] 高质量RNA的提取是定量逆转录PCR（qRT-PCR）和微阵列分析的必要步骤。 然而，从含有大量多糖，脂质和次级代谢物的水果材料中提取高质量RNA是不容易的。 Wan和Wilkins（1994）开发了"热硼酸盐方法"来分离高质量的RNA。 在这里，我们描述了"热硼酸盐方法"的修饰的协议，从葡萄浆果皮肤中分离高质量的RNA用于qRT-PCR和微阵列分析（Suzuki等人，2015a; Suzuki等人 al。，2015b）。
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| Mitochondrial RNA Transcript Analysis Assay of Arabidopsis Leaf Tissues
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Author:
Date:
2015-10-20
[Abstract] This qPCR-based assay provides an overview of the expression levels of all mitochondrial transcripts (mRNAs and rRNAs) as well as splicing efficiency in Arabidopsis. It was developed before RNAseq techniques were widely used (de Longevialle et al., 2007), but is nevertheless still useful as it is cheaper to run and the analysis is much easier and faster to perform if the aim is only to look at mitochondrial transcripts. For intron-containing mRNAs, the use of primer sets specifically amplifying spliced or unspliced forms allows the evaluation of the splicing efficiency.
[摘要] 这种基于qPCR的测定提供了所有线粒体转录物（mRNA和rRNA）的表达水平以及拟南芥中的剪接效率的概述。 它是在RNAseq技术被广泛使用之前开发的（de Longevialle等人，2007），但是仍然有用，因为它运行更便宜，并且分析更容易和更快地执行，如果目标 只是看看线粒体转录物。 对于含内含子的mRNA，使用特异性扩增剪接或未剪接形式的引物组允许评价剪接效率。
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| CytoTrap Two-Hybrid Screening Assay
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Author:
Date:
2014-12-05
[Abstract] CytoTrap two-hybrid system provides an alternate strategy to detect protein-protein interactions in yeast. In this system, bait protein is fused with human son of sevenless (hSos) protein (Li et al., 1993), and a cDNA library or prey protein is expressed by fusion with myristoylation signal which anchors the prey fusion protein to yeast cell membrane. Protein interaction between bait and prey proteins recruits the hSos protein to the cell membrane, where hSos activates the Ras signaling pathway, leading to the survival of temperature-sensitive Saccharomyces cerevisiae (S. ...
[摘要] CytoTrap双杂交系统提供了检测酵母中蛋白质 - 蛋白质相互作用的替代策略。在该系统中，诱饵蛋白与七（hSos）蛋白质的人子融合（Li等，1993），cDNA文库或猎物蛋白质与肉豆蔻酰化信号的融合表达，将猎物融合蛋白锚定到酵母细胞膜。诱饵和猎物蛋白之间的蛋白质相互作用将hSos蛋白质募集到细胞膜上，其中hSos激活Ras信号通路，导致温度敏感的酿酒酵母（S.cerevisiae）菌株cdc25H在36℃下的存活。在CytoTrap双杂交系统中，蛋白质相互作用的检测发生在细胞膜附近的细胞质中，不依赖于报告基因的转录激活。因此，该系统对于识别需要在细胞质中进行翻译后修饰的转录因子和蛋白质的相互作用伴侣特别有用，其在常规基于反式激活的酵母双杂交系统中不能用作诱饵蛋白。在这里，我们描述了来自模拟植物拟南芥的cDNA文库的构建以及用于筛选来自该文库的AtSR1 / CAMTA3，Ca2 + / CaM调节的转录因子的相互作用蛋白的程序。该过程可以适应于识别来自其他生物体的感兴趣的蛋白质的相互作用的伴侣。
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