Note Fluorescence activated cell sorting (FACS) using RNAlater to minimize RNA degradation and perturbation of mRNA expression from cells involved in initial host microbe interactions Kevin P. Nishimoto a , Daniel Newkirk b , Stephen Hou c , John Fruehauf c , Edward L. Nelson a,c,d, ⁎ a Molecular Biology and Biochemistry, School of Biological Sciences, University of California, Irvine, Irvine, CA, USA b DNA & Protein MicroArray Facility, Department of Biological Chemistry, University of California, Irvine, Irvine, CA, USA c Department of Medicine, Division of Hematology/Oncology, School of Medicine, University of California, Irvine, Irvine, CA, USA d Center for Immunology, University of California, Irvine, Irvine, CA, USA Received 15 January 2007; received in revised form 27 March 2007; accepted 27 March 2007 Available online 12 April 2007 Abstract Host microbe interactions frequently involve specific cellular tropism. Accurate characterization of cells involved in these initial interactions is complicated by the response to the microbe. We describe a method utilizing RNAlater for Fluorescence Activated Cell Sorting of these critical cells that minimizes the downstream perturbation in the gene expression profile. © 2007 Elsevier B.V. All rights reserved. Keywords: Fluorescence activated cell sorting; Host microbe interactions; Gene expression profile; RNA It is well known foreign pathogens and microbes have a selective tropism for particular tissues or cells and that host cells can respond very quickly to interactions with microbes. The accurate characterization of these cells targeted by microbes is important for understanding pathopysiologic mechanisms and pathogen host interactions. Unfortunately, methods to isolate RNA from un-perturbed cellular subsets for expression profiling is frequently complicated by activation of cells due to the induced microbe response, which can lead to altered RNA expression patterns (Arbibe et al., 2007) and to potential RNA degradation during the employed isolation procedure. Fluorescence activated cell sorting (FACS) is a convenient and efficient method for isolating cells of interest that interact with potential microbes. This procedure can be partially con- ducted at 4 °C, however methods to minimize cellular activation or perturbation during the entire FACS procedure for accurate RNA analyses have not been reported. It is widely recognized that cells undergoing the FACS process can experience physiologic stress and even a decrease in viability. Several external factors associated with cell sorting such as flow rate, pressure, and droplet charge, may potentially cause modulations in cellular physiology including RNA transcript levels for cells that are sensitive to these processes. Altered RNA expression patterns may become further exacerbated due to altered conditions that cells experience when streamed into collection tubes containing buffered solutions, such as culture medium or PBS, the accompanying change in temperature, and the time that the cells are in this solution until the process is complete. Therefore, the combination of the host cell response to the interacting microbe and external factors associated with cell- sorting may lead to inaccurate RNA expression profiles of the isolated cell population. Several potential options exist including immediate cell lysis and protein denaturization, e.g. the commonly used Trizol reagent (Invitrogen, Carlsbad, CA). Journal of Microbiological Methods 70 (2007) 205 – 208 www.elsevier.com/locate/jmicmeth Abbreviations: FACS, Fluorescence activated cell sorting; PBMCs, Peripheral blood mononuclear cells; VRP, Venezuelan Encephalitis virus derived Replicon Particle. ⁎ Corresponding author. University of California at Irvine, Hewitt Hall, Irvine, CA 92697, USA. Tel.: +1 949 824 2860; fax: +1 949 824 2305. E-mail address: enelson@uci.edu (E.L. Nelson). 0167-7012/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2007.03.022