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Trizol

Company: Invitrogen
Catalog#: 15596018
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Using RNA Sequencing and Spike-in RNAs to Measure Intracellular Abundance of lncRNAs and mRNAs
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 ...

Flow Cytometry Analysis and Fluorescence-activated Cell Sorting of Myeloid Cells from Lung and Bronchoalveolar Lavage Samples from Mycobacterium tuberculosis-infected Mice
Author:
Date:
2020-05-20
[Abstract]  Mycobacterium tuberculosis (Mtb) is transmitted by aerosol and can cause serious bacterial infection in the lung that can be fatal if left untreated. Mtb is now the leading cause of death worldwide by an infectious agent. Characterizing the early events of in vivo infection following aerosol challenge is critical for understanding how innate immune cells respond to infection but is technically challenging due to the small number of bacteria that initially infect the lung. Previous studies either evaluated Mtb-infected cells at later stages of infection when the number of bacteria in the lung is much higher or used in vitro model systems to assess the response of myeloid cells to Mtb. Here, we describe a method that uses fluorescent bacteria, a high-dose aerosol ... [摘要]  [摘要 ] 结核分枝杆菌(Mtb)通过气溶胶传播,可引起严重的肺部细菌感染,如果不及时治疗,可能致命。Mtb现在已成为全球传染病致死的主要原因。表征气溶胶激发后体内感染的早期事件对于了解先天免疫细胞如何对感染做出反应至关重要,但由于最初会感染肺的细菌数量少,因此在技术上具有挑战性。先前的研究或者在肺部细菌数量高得多时在感染后期评估Mtb感染的细胞,或者在体外使用 评估骨髓细胞对Mtb反应的模型系统。在这里,我们介绍一种使用荧光细菌,大剂量气溶胶感染模型和流式细胞术跟踪气溶胶感染和荧光激活细胞分选(FACS)之后立即分离肺中Mtb感染细胞的方法,以分离幼稚的旁观者,和Mtb感染的细胞用于下游应用,包括RNA测序。该协议提供了在肺环境中监视Mtb感染和细胞特异性反应的能力,已知该环境可调节常驻和募集人群的功能。使用此协议,我们发现肺泡巨噬细胞通过上调受转录因子Nrf2调节并有害于细菌早期控制的细胞保护性转录反应,在体内对Mtb感染作出反应。

[背景 ] 气溶胶传播是结核分枝杆菌(Mtb)感染自然周期的关键组成部分,有助于细菌的毒性并导致其在肺部的独特感染模式(North ,1995;Riley 等,1995)。 ; Pai et ...

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