Isolation of intact RNA from murine CD4 + T cells after intracellular cytokine staining and fluorescence-activated cell sorting

Shajo Kunnath Velayudhan, Steven A. Porcelli

Research output: Contribution to journalArticle

2 Scopus citations

Abstract

Intracellular cytokine staining (ICS) is a powerful method for identifying functionally distinct lymphocyte subsets, and for isolating these by fluorescence activated cell sorting (FACS). Although transcriptomic analysis of cells sorted on the basis of ICS has many potential applications, this is rarely performed because of the difficulty in isolating intact RNA from cells processed using standard fixation and permeabilization buffers for ICS. To address this issue, we compared three buffers shown previously to preserve RNA in nonhematopoietic cells subjected to intracellular staining for their effects on RNA isolated from T lymphocytes processed for ICS. Our results showed that buffers containing the recombinant ribonuclease inhibitor RNasin or high molar concentrations of salt yielded intact RNA from fixed and permeabilized T cells. As proof of principle, we successfully used the buffer containing RNasin to isolate intact RNA from CD4 + T cells that were sorted by FACS on the basis of specific cytokine production, thus demonstrating the potential of this approach for coupling ICS with transcriptomic analysis.

Original languageEnglish (US)
Pages (from-to)77-80
Number of pages4
JournalJournal of Immunological Methods
Volume456
DOIs
StatePublished - May 2018

Keywords

  • FACS
  • Intracellular cytokine staining
  • RNA isolation
  • T cells
  • Transcriptomics

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

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