Measurement of Low‐Abundance Intracellular mRNA Using Amplified FISH Staining and Image‐Based Flow Cytometry

Q1 Health Professions

Current Protocols in Cytometry Pub Date : 2016-04-01 DOI:10.1002/0471142956.cy0746s76

A. L. Henning, J. N. B. Sampson, B. McFarlin

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引用次数: 13

Abstract

Recent advances in instrument design and reagent development have enabled the rapid progression in available measurement techniques in the field of flow cytometry. In particular, image‐based flow cytometry extends the analysis capacity found in traditional flow cytometry. Until recently, it was not possible to measure intracellular mRNA in specific phenotypes of cells by flow cytometry. In this protocol, a method of completing simultaneous intracellular measurement of mRNA and protein for PPAR‐gamma in peripheral blood monocytes, which have been exposed in vitro to modified LDL, is described. The process of PPAR‐gamma activation following uptake of modified LDL is believed to play a role in the development of atherogenesis. PPAR‐gamma mRNA measurement was made possible using an amplified FISH technique (PrimeFlow RNA Assay) that allowed for detection of low‐abundant intracellular mRNA expression. This protocol represents a continued effort by the authors’ laboratory to establish and validate new techniques to assess the role of the immune system in chronic disease. © 2016 by John Wiley & Sons, Inc.

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使用扩增的FISH染色和基于图像的流式细胞术测量细胞内低丰度mRNA

仪器设计和试剂开发的最新进展使流式细胞术领域的可用测量技术迅速发展。特别是，基于图像的流式细胞术扩展了传统流式细胞术的分析能力。直到最近，还不可能通过流式细胞术测量细胞内特定表型细胞的mRNA。在本方案中，描述了一种在体外暴露于修饰LDL的外周血单核细胞中完成PPAR γ mRNA和蛋白质的细胞内同步测量的方法。摄取修饰LDL后的PPAR - γ激活过程被认为在动脉粥样硬化的发展中起作用。使用扩增的FISH技术(PrimeFlow RNA Assay)可以检测低丰度的细胞内mRNA表达，从而可以测量PPAR - γ mRNA。该方案代表了作者实验室建立和验证新技术以评估免疫系统在慢性疾病中的作用的持续努力。©2016 by John Wiley & Sons, Inc。

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Current Protocols in Cytometry Health Professions-Medical Laboratory Technology

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期刊介绍： Published in affiliation with the International Society for Advancement of Cytometry, Current Protocols in Cytometry is a "best practices" collection that distills and organizes the absolute latest techniques from the top cytometry labs and specialists worldwide. It is the most complete set of peer-reviewed protocols for flow and image cytometry available.