title: High salt buffer improves integrity of RNA after fluorescence-activated cell sorting of intracellular labeled cells.
abstract: Over the past years, massive progress has been made in the ability to collect large-scale gene expression data from a limited sample size. Combined with improvements in multiplex flow cytometry-based techniques, this has made it possible to isolate and characterize specific cellular subtypes within heterogeneous populations, with a great impact on our understanding of different biological processes. However, sorting based on intracellular markers requires fixation and permeabilization of samples, and very often the integrity of RNA molecules is compromised during this process. Many attempts have been made to improve the quality of nucleic acids from such samples, but RNA degradation still remains a limiting factor for downstream analyses. Here we present a method to isolate high quality RNA from cells that have been fixed, permeabilized, intracellularly labeled and sorted. By performing all incubation steps in the presence of a high salt buffer, RNA degradation was avoided and samples with remarkable integrity were obtained. This procedure offers a straightforward and very affordable technique to retrieve high quality RNA from isolated cell populations, which increases the possibilities to characterize gene expression profiles of subpopulations from mixed samples, a technique with implications in a broad range of research fields.
authors.affiliation: Department of Laboratory Medicine, Center for Molecular Pathology, Lund University, Sweden. Electronic address: helen.nilsson@med.lu.se.
authors.forename: Helén
authors.initials: H
authors.lastname: Nilsson
authors.affiliation: Department of Laboratory Medicine, Center for Molecular Pathology, Lund University, Sweden.
authors.forename: Krzysztof M
authors.initials: KM
authors.lastname: Krawczyk
authors.affiliation: Department of Laboratory Medicine, Center for Molecular Pathology, Lund University, Sweden.
authors.forename: Martin E
authors.initials: ME
authors.lastname: Johansson
mesh_terms: D000906:Antibodies / Q000494:pharmacology; D002021:Buffers; D002232:Carbocyanines / Q000494:pharmacology; D002460:Cell Line; D045744:Cell Line, Tumor; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000494:pharmacology; D005557:Formaldehyde / Q000494:pharmacology; D006801:Humans; D053552:Keratin-7 / Q000276:immunology; D053533:Keratin-8 / Q000276:immunology; D000432:Methanol / Q000494:pharmacology; D011108:Polymers / Q000494:pharmacology; D012313:RNA / Q000302:isolation & purification*; D020871:RNA Stability / Q000187:drug effects*; D012965:Sodium Chloride / Q000494:pharmacology*; D013194:Staining and Labeling / Q000379:methods*
chemical_list: C569686:Alexa Fluor 647; D000906:Antibodies; D002021:Buffers; D002232:Carbocyanines; D005456:Fluorescent Dyes; D053552:Keratin-7; D053533:Keratin-8; D011108:Polymers; D005557:Formaldehyde; D012965:Sodium Chloride; D012313:RNA; C003043:paraform; D000432:Methanol
keywords: Fixation; Fluorescence-activated cell sorting; Intracellular antibody labeling; NaCl; RNA integrity