Abstract
The protocol described here has been developed to detect RNA at the single cell level. Fluorescent probes hybridize to target RNAs and are detected by flow cytometry after multiple amplification steps. Different types of RNA can be detected such as mRNA, long noncoding RNA, viral RNA or telomere RNA and up to 4 different target probes can be used simultaneously. We used this protocol to specifically measure the expression of two transcription factor mRNAs, MAFB and IRF4, in human monocytes.
Keywords: RNA, Flow cytometry, In situ, Hybridization, Single cell, Human
Background
RT-qPCR is one major technique used to easily assess RNA expression. Cells are lysed and analyzed in bulk. Hence, cell heterogeneity is lost. In particular, it is impossible using RT-qPCR to address whether subpopulations may be identified based on the expression of RNA. RNA Fluorescent In Situ Hybridization (FISH) is a way to detect RNA in single cells. This technique requires hybridization of fluorescent probes on RNA targets that are then detected using an imaging system such as a confocal microscope. However, this method is time-consuming and allows the analysis of a limited number of individual cells. We demonstrated by RT-qPCR that human monocytes cultured in RPMI with M-CSF, IL-4, and TNFa express after three hours the transcription factors MAFB and IRF4 (Goudot et al., 2017). MAFB and IRF4 are involved in the differentiation of monocytes into monocyte-derived macrophages (mo-mac) and monocyte-derived DC (mo-DC) respectively. To decipher whether monocytes express both transcription factors or if this expression is mutually exclusive, we performed in situ hybridization coupled to flow cytometry using PrimeFlow RNA assay.
Materials and Reagents
Equipment
Software
Procedure
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Data analysis
Data analysis was performed with FlowJo V10. Single color tubes were used to calculate the compensation matrix. Monocytes were cultured with M-CSF only (that drives mo-mac differentiation) or with a cocktail of M-CSF, IL-4, TNF-a and FICZ (that induces monocytes to differentiate preferentially into mo-DC). We found that a part of monocytes express MAFB mRNA and others IRF4 mRNA (Figure 1). No monocyte expressed the two mRNA simultaneously (Figure 1). PrimeFlow RNA assay showed that monocytes cultured for 3 h with M-CSF, IL-4, TNFa and FICZ express MAFB or IRF4 and that their expression levels are mutually exclusive (Goudot et al., 2017). Figure 1. Proportions of MAFB+ and IRF4+ monocytes at 3 h and 12 h. Monocytes were subjected to PrimeFlow RNA assay directly after isolation or after 3 h or 12 h of culture with M-CSF or with M-CSF, IL-4, TNFa and FICZ. AlexaFluor488-MAFB and AlexaFluor647-IRF4 probes were used. A. Gating strategy. Debris and dead cells were gated out. B. MAFB mRNA and IRF4 mRNA expression.
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Acknowledgments
This work was supported by Agence Nationale de la Recherche (program “Investissements d’Avenir” ANR-10-LABX-0043, ANR-10-IDEX-0001-02 PSL, and ANR-17-CE15-0011-01), INSERM and Fondation Bristol-Myers Squibb pour la Recherche en Immuno-Oncologie.
Competing interests
Authors declare no conflict of interest.
References
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