| 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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| HBV Infection in Human Hepatocytes and Quantification of Encapsidated HBV DNA
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Author:
Date:
2016-01-20
[Abstract] Human hepatic cancer cell lines such as HepG2, Huh7, and HLE cannot get infected with Hepatitis B virus (HBV) due to lack of an HBV receptor(s). Transfection with HBV genome has so far been referred as a tool to mimic HBV infection. However, since sodium taurocholate cotransporting polypeptide (NTCP) was identified as a functional receptor for HBV (Yan et al., 2012), hepatocyte cell lines that were stably transfected with a plasmid for NTCP expression have been used for HBV infection. This protocol is designed for infection with HBV in human hepatocyte cell line HepG2 expressing NTCP (HepG2-hNTCP-C4 cells; Iwamoto et al., 2014) or primary human hepatocytes (PHHs). In this section, we also describe one of the methods for the assessment of HBV infection: Quantification of ...
[摘要] 人肝癌细胞系如HepG2，Huh7和HLE由于缺乏HBV受体而不能感染乙型肝炎病毒（HBV）。 HBV基因组的转染迄今为止被称为模拟HBV感染的工具。 然而，由于牛磺胆酸钠共转运多肽（NTCP）被鉴定为HBV的功能性受体（Yan等人，2012），已经使用用用于NTCP表达的质粒稳定转染的肝细胞细胞系 为HBV感染。 该方案设计用于在表达人类肝细胞细胞系HepG2的表达NTCP（HepG2-hNTCP-C4细胞; Iwamoto等人，2014）或原代人肝细胞（PHH）的HBV感染。 在本节中，我们还描述了用于评估HBV感染的方法之一：细胞内衣壳化的HBV DNA的定量。
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