title: Simultaneous detection of mRNA and protein in single cells using immunofluorescence-combined single-molecule RNA FISH.
abstract: Although the concept of combining immunofluorescence (IF) with single-molecule RNA fluorescence in situ hybridization (smRNA FISH) seems obvious, the specific materials used during IF and smRNA FISH make it difficult to perform these procedures simultaneously on the same specimen. Even though there are reports where IF and smRNA FISH were combined with success, these were insufficient in terms of signal intensities, staining patterns, and GFP-compatibility, and a detailed exploration of the various factors that influence IF and smRNA FISH outcome has not been published yet. Here, we report a detailed study of conditions and reagents used in classic IF and smRNA FISH that allowed us to establish an easy, robust, and GFP-compatible procedure. Our protocol enables simultaneous detection of mRNA and protein quantity as well as the subcellular distribution of these molecules in single cells by combining an RNase-free modification of the IF technique and the more recent smRNA FISH method. Using this procedure, we have shown the direct interaction of RNase MCPIP1 with IL-6 mRNA. We also demonstrate the use of our protocol in heterogeneous cell population analysis, revealing cell-to-cell differences in mRNA and protein content.
authors.affiliation: Department of Cell Biochemistry, Faculty of Biochemistry, Biophysics, and Biotechnology, Jagiellonian University, Cracow, Poland.
authors.forename: Jakub
authors.initials: J
authors.lastname: Kochan
authors.affiliation: Department of Cell Biochemistry, Faculty of Biochemistry, Biophysics, and Biotechnology, Jagiellonian University, Cracow, Poland.
authors.forename: Mateusz
authors.initials: M
authors.lastname: Wawro
authors.affiliation: Department of Cell Biochemistry, Faculty of Biochemistry, Biophysics, and Biotechnology, Jagiellonian University, Cracow, Poland.
authors.forename: Aneta
authors.initials: A
authors.lastname: Kasza
mesh_terms: D005455:Fluorescent Antibody Technique / Q000379:methods; D006367:HeLa Cells; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism; D036103:Nanotechnology; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification*; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification*; D012260:Ribonucleases / Q000235:genetics / Q000378:metabolism; D059010:Single-Cell Analysis; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D015850:Interleukin-6; D011506:Proteins; D012333:RNA, Messenger; D014157:Transcription Factors; D012260:Ribonucleases; C510646:ZC3H12A protein, human
keywords: MCPIP1; RNA; immunofluorescence; single-cell analysis; smRNA FISH