|
Author:
Date:
2021-04-20
[Abstract] Hepatitis B virus (HBV) is the major cause of liver diseases and liver cancer worldwide. After infecting hepatocytes, the virus establishes a stable episome (covalently closed circular DNA, or cccDNA) that serves as the template for all viral transcripts. Specific and accurate quantification of cccDNA is difficult because infected cells contain abundant replicative intermediates of HBV DNA that share overlapping sequences but arranged in slightly different forms. HBV cccDNA can be detected by Southern blot or qPCR methods which involve enzymatic digestion. These assays are laborious, have limited sensitivity, or require degradation of cellular DNA (which precludes simple normalization). The method described in this protocol, cccDNA inversion quantitative (cinq)PCR, instead uses a series ...
[摘要] [摘要]乙型肝炎病毒（HBV）是全球范围内肝脏疾病和肝癌的主要原因。感染肝细胞后，病毒建立起稳定的附加体（共价闭合的环状DNA或cccDNA），作为所有病毒转录本的模板。cccDNA的特异性和准确定量非常困难，因为被感染的细胞含有丰富的HBV DNA复制中间体，这些中间体共享重叠序列，但排列形式略有不同。HBV cccDNA可以通过涉及酶消化的Southern印迹或qPCR方法进行检测。这些测定费力，灵敏度有限或需要细胞DNA降解（无法进行简单的标准化）。该协议中描述的方法cccDNA反向定量（cinq）PCR，而是使用一系列限制性酶介导的水解和连接反应，将cccDNA转化为反向线性扩增子，该扩增子无法从其他形式的HBV DNA扩增或检测到。重要的是，细胞DNA在样品制备过程中仍可定量，从而可以进行标准化并显着提高精确度。另外，第二线性片段（源自酶消化HBV DNA基因组的单独区域，并以所有形式的HBV DNA存在）可用于同时定量总HBV水平。
图形摘要：
HBV的cccDNA和总HBV DNA的选择性检测使用cinqPCR （转载自涂等人，2020一）。
[背景]乙型肝炎病毒（HBV）是一种小的有包膜病毒，其encapsidates一个部分双链环状DNA基因组，所谓松弛环状（RC）的DNA。感染人肝细胞后，核衣壳被转运至细胞核，其中rcDNA基因组被转化为共价闭合的环状（ccc）DNA。这种游离形式是高度稳定的，并保持慢性HBV感染（Tu等人，2020b ...
|
|
Author:
Date:
2020-12-20
[Abstract] The mRNA therapeutics is a new class of medicine to treat many various diseases. However, in vitro transcribed (IVT) mRNA triggers immune responses due to recognition by human endosomal and cytoplasmic RNA sensors, but incorporation of modified nucleosides have been shown to reduce such responses. Therefore, an assay signifying important aspects of the human immune system is still required. Here, we present a simple ex vivo method called ‘RNA ImmunoGenic Assay’ to measure immunogenicity of IVT-mRNAs in human whole blood. Chemically modified and unmodified mRNA are complexed with a transfection reagent (TransIT), and co-incubated in human whole blood. Specific cytokines are measured (TNF-α, INF-α, INF-γ, IL-6 and IL-12p70) using ELISAs. The ...
[摘要] [摘要] mRNA疗法是治疗多种疾病的新型药物。然而，我Ñ体外转录（IVT）的mRNA触发由于人类内体和细胞质RNA传感器，但是修饰的核苷的掺入识别的免疫应答已经示出吨ö减少此类反应。牛逼herefore ，测定标志着重要的环节人体免疫系统仍然需要。这里，我们提出一个简单的离体称为“RNA方法免疫ģ ENIC测定”测量的IVT-mRNA的免疫原性小号在人全血。将化学修饰和未修饰的mRNA与转染试剂（TransIT ）复合，并在人全血中共同孵育。特异性细胞因子测定（TNF- α ，INF- α ，INF- γ ，IL-6和IL-12p70的），使用的ELISA。进行qPCR分析以揭示特异性免疫途径的激活。所述RNA免疫ģ ENIC测定提供小号的简单且快速的方法来检测供体特异性-针对mRNA的治疗剂的免疫应答。
图形摘要：
RNA免疫基因测定的示意图
[背景] mRNA治疗是基因治疗的重要一类（Sahin等，2014 ；Antony等，2015 ...
|