| Using RNA Sequencing and Spike-in RNAs to Measure Intracellular Abundance of lncRNAs and mRNAs
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Author:
Date:
2020-10-05
[Abstract] Long noncoding RNAs (lncRNAs) play essential roles in normal physiology and in disease but their mechanisms of action can be challenging to identify. For mechanistic studies, it is often useful to know a lncRNA’s intracellular abundance, i.e., approximately how many molecules of the lncRNA are present in a typical cell of a cell-type of interest. At least two approaches have been used to approximate lncRNA intracellular abundance: single-molecule sensitivity RNA fluorescence in situ hybridization (smFISH) and single-gene, calibrated reverse-transcription followed by quantitative PCR (RT-qPCR). However, like all experimental approaches, these methods have their limitations. smFISH, when analyzed using diffraction-limited microscopy, can underestimate intracellular ...
[摘要] [摘要]长非编码RNA（lncRNA）在正常生理和疾病中起着至关重要的作用，但其作用机理可能难以鉴定。对于机理研究，了解lncRNA的胞内丰度（即在目标细胞类型的典型细胞中大约存在多少个lncRNA分子）通常很有用。至少两种方法已用于估算lncRNA细胞内丰度：单分子敏感性RNA荧光原位杂交（smFISH ...
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| Isolation and Quantification of Extracellular DNA from Biofluids
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Author:
Date:
2020-08-20
[Abstract] Extracellular DNA is studied as a diagnostic biomarker, but also as a factor involved in the pathophysiology of several diseases due to its pro-inflammatory properties. Extracellular DNA can be extracted from plasma, urine, saliva or other biofluids using standard DNA isolation procedures and specialized commercial kits. Sample preparation for isolation is important, freezing and thawing may affect the amount of extracellular DNA extracted. Subsequent centrifugations remove cells and cell debris from the samples to obtain true extracellular DNA. Small volume of samples especially from animal experiments is often an issue and it affects the DNA yield. Very short fragments (˂ 100 bp) can be lost during isolation and are difficult to quantify using PCR. Fluorometric methods asses all stained ...
[摘要] [摘要 ] 研究细胞外DNA作为一种诊断性生物标志物，但由于其具有促炎性，也可以作为多种疾病的病理生理因素。可以使用标准DNA分离程序和专门的商业试剂盒从血浆，尿液，唾液或其他生物流体中提取细胞外DNA。样品制备对于分离非常重要，冷冻和解冻可能会影响提取的细胞外DNA的量。随后的离心去除样品中的细胞和细胞碎片，以获得真正的细胞外DNA。少量样品尤其是动物实验样品常常是一个问题，它会影响DNA产量。片段很短（˂ 100 bp）在分离过程中可能会丢失，并且难以使用PCR进行定量。荧光法评估所有染色的DNA片段。选择定量细胞外DNA的方法至关重要，并且至少两种方法的组合是理想的。程序的标准化或至少在研究论文中的报告对于比较结果至关重要。
[背景 ] 胞外DNA通常被称为无细胞DNA是所有DNA的一个术语发现特别是在诊断生物流体细胞外。血浆DNA最早是由Mandel和Metais （1948）发现的。后来人们对所谓的液体活检的研究激发了人们对细胞外DNA研究的兴趣，液体活检作为无创筛选和诊断的基于DNA的生物标志物的来源（Poon和Lo，2001）。相同的胞外DNA，然而，也参与炎性疾病，如败血症的病理生理学（Lauková 等人，2017） ，急性肾损伤（詹森等人，2017）和急性肝衰竭（Vokálová ...
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| Generation of Fusarium graminearum Knockout Mutants by the Split-marker Recombination Approach
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Author:
Date:
2018-08-20
[Abstract] Fusarium graminearum is a destructive phytopathogen and shows an impressive metabolic diversity. Gene deletion is an important and useful approach for gene function study. Here we present a protocol for generating gene deletion mutant by applying “split-marker” deletion strategy (Catlett et al., 2003) with PEG-mediated protoplast transformation (Yuan et al., 2008; Martín, 2015).
[摘要] 禾谷镰刀菌是一种破坏性的植物病原体，具有令人印象深刻的代谢多样性。 基因缺失是基因功能研究的重要且有用的方法。 在这里，我们提出了一个协议，通过应用“分裂标记”删除策略（Catlett et al。，2003）与PEG介导的原生质体转化（Yuan 等。，2008;Martín，2015）。
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