| RI-SEC-seq: Comprehensive Profiling of Nonvesicular Extracellular RNAs with Different Stabilities
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Author:
Date:
2021-02-20
[Abstract] Exosomes and other extracellular vesicles (EVs) are considered the main vehicles transporting RNAs in extracellular samples, including human bodily fluids. However, a major proportion of extracellular RNAs (exRNAs) do not copurify with EVs and remain in ultracentrifugation supernatants of cell-conditioned medium or blood serum. We have observed that nonvesicular exRNA profiles are highly biased toward those RNAs with intrinsic resistance to extracellular ribonucleases. These highly resistant exRNAs are interesting from a biomarker point of view, but are not representative of the actual bulk of RNAs released to the extracellular space. In order to understand exRNA dynamics and capture both stable and unstable RNAs, we developed a method based on size-exclusion chromatography (SEC) ...
[摘要] [摘要]外来体和其他细胞外囊泡（EVs）被认为是在细胞外样品（包括人体液）中运输RNA的主要载体。但是，大部分细胞外RNA（exRNA ）不能与EV共纯化，而是保留在细胞条件培养基或血清的超速离心上清液中。我们已经观察到非囊泡的exRNA概况高度偏向那些对细胞外核糖核酸酶具有固有抗性的RNA。从生物标志物的角度来看，这些高度抗性的exRNA很有趣，但不能代表释放到细胞外空间的RNA的实际体积。为了了解exRNA动态并捕获稳定和不稳定的RNA，我们开发了一种基于大小排阻色谱（SEC）分馏的RNase抑制剂（RI）处理的细胞条件培养基（RI-SEC-seq）的方法。这种方法使我们能够鉴定和研究细胞外核糖体和tRNA，并提供了可以在不久的将来影响生物标志物发现的细胞外RNAome的动态视图。
图形概要：
所述RI-SEC-SEQ协议的概述：大小排阻层析的级分的测序从nonvesicular胞样品用或不用RNA酶抑制剂（+/- RI）
[背景]细胞外RNA（exRNA ）参与细胞间通讯，并且在微创液体活检中有望成为疾病的生物标志物（O'Brien et ...
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| RNA Isolation and Northern Blot Analysis
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Author:
Date:
2014-03-20
[Abstract] The northern blot is a technique used in molecular biology research to study gene expression by detection of RNA in a sample. With northern blotting it is possible to observe particular gene expression levels during differentiation, morphogenesis, as well as abnormal or diseased conditions. Here, we examine ATF3, ATF4, and GADD153 gene expression profiles by northern blot in Vero cells and H1299 cells after IBV infection. RNA was extracted in IBV (infectious bronchitis virus) infected cells and electrophoresis was used to separate the RNA sample. RNA was transferred from the electrophoresis gel to the blotting membrane by capillary transfer. Specific mRNA was detected with hybridization probes complementary to part of target sequence. The probes were prepared by RT-PCR and labeled by ...
[摘要] Northern印迹是在分子生物学研究中用于通过检测样品中的RNA来研究基因表达的技术。 使用Northern印迹，可以在分化，形态发生以及异常或疾病状况期间观察到特定的基因表达水平。 在这里，我们检查ATF3，ATF4和GADD153基因表达谱通过北部污点在Vero细胞和H1299细胞后IBV感染。 在IBV（感染性支气管炎病毒）感染的细胞中提取RNA，并使用电泳分离RNA样品。 通过毛细管转移将RNA从电泳凝胶转移到印迹膜上。 使用与靶序列的一部分互补的杂交探针检测特异性mRNA。 通过RT-PCR制备探针，并使用DIG标记试剂盒通过地高辛（DIG）标记。
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