Simultaneous detection of mRNA and protein in single cells using immunofluorescence-combined single-molecule RNA FISH
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...
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Taylor & Francis Group
2015-10-01
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| Series: | BioTechniques |
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| Online Access: | https://www.future-science.com/doi/10.2144/000114340 |
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| author | Jakub Kochan Mateusz Wawro Aneta Kasza |
| author_facet | Jakub Kochan Mateusz Wawro Aneta Kasza |
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| description | 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. |
| format | Article |
| id | doaj-art-c1d5d50e424c43dc9415268d43d4c8a8 |
| institution | OA Journals |
| issn | 0736-6205 1940-9818 |
| language | English |
| publishDate | 2015-10-01 |
| publisher | Taylor & Francis Group |
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| series | BioTechniques |
| spelling | doaj-art-c1d5d50e424c43dc9415268d43d4c8a82025-08-20T02:25:55ZengTaylor & Francis GroupBioTechniques0736-62051940-98182015-10-0159420922110.2144/000114340Simultaneous detection of mRNA and protein in single cells using immunofluorescence-combined single-molecule RNA FISHJakub Kochan0Mateusz Wawro1Aneta Kasza21Department of Cell Biochemistry, Faculty of Biochemistry, Biophysics, and Biotechnology, Jagiellonian University, Cracow, Poland1Department of Cell Biochemistry, Faculty of Biochemistry, Biophysics, and Biotechnology, Jagiellonian University, Cracow, Poland1Department of Cell Biochemistry, Faculty of Biochemistry, Biophysics, and Biotechnology, Jagiellonian University, Cracow, PolandAlthough 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.https://www.future-science.com/doi/10.2144/000114340immunofluorescencesmRNA FISHRNAsingle-cell analysisMCPIP1 |
| spellingShingle | Jakub Kochan Mateusz Wawro Aneta Kasza Simultaneous detection of mRNA and protein in single cells using immunofluorescence-combined single-molecule RNA FISH BioTechniques immunofluorescence smRNA FISH RNA single-cell analysis MCPIP1 |
| title | Simultaneous detection of mRNA and protein in single cells using immunofluorescence-combined single-molecule RNA FISH |
| title_full | Simultaneous detection of mRNA and protein in single cells using immunofluorescence-combined single-molecule RNA FISH |
| title_fullStr | Simultaneous detection of mRNA and protein in single cells using immunofluorescence-combined single-molecule RNA FISH |
| title_full_unstemmed | Simultaneous detection of mRNA and protein in single cells using immunofluorescence-combined single-molecule RNA FISH |
| title_short | Simultaneous detection of mRNA and protein in single cells using immunofluorescence-combined single-molecule RNA FISH |
| title_sort | simultaneous detection of mrna and protein in single cells using immunofluorescence combined single molecule rna fish |
| topic | immunofluorescence smRNA FISH RNA single-cell analysis MCPIP1 |
| url | https://www.future-science.com/doi/10.2144/000114340 |
| work_keys_str_mv | AT jakubkochan simultaneousdetectionofmrnaandproteininsinglecellsusingimmunofluorescencecombinedsinglemoleculernafish AT mateuszwawro simultaneousdetectionofmrnaandproteininsinglecellsusingimmunofluorescencecombinedsinglemoleculernafish AT anetakasza simultaneousdetectionofmrnaandproteininsinglecellsusingimmunofluorescencecombinedsinglemoleculernafish |