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Simultaneous visualization of RNA transcripts and proteins in whole-mount mouse preimplantation embryos using single-molecule fluorescence in situ hybridization and immunofluorescence microscopy
Whole-mount single-molecule RNA fluorescence in situ hybridization (smRNA FISH) in combination with immunofluorescence (IF) offers great potential to study long non-coding RNAs (lncRNAs): their subcellular localization, their interactions with proteins, and their function. Here, we describe a step-b...
Autores principales: | , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Frontiers Media S.A.
2022
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9577017/ https://www.ncbi.nlm.nih.gov/pubmed/36268512 http://dx.doi.org/10.3389/fcell.2022.986261 |
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author | Hazra, Rasmani Spector, David L. |
author_facet | Hazra, Rasmani Spector, David L. |
author_sort | Hazra, Rasmani |
collection | PubMed |
description | Whole-mount single-molecule RNA fluorescence in situ hybridization (smRNA FISH) in combination with immunofluorescence (IF) offers great potential to study long non-coding RNAs (lncRNAs): their subcellular localization, their interactions with proteins, and their function. Here, we describe a step-by-step, optimized, and robust protocol that allows detection of multiple RNA transcripts and protein molecules in whole-mount preimplantation mouse embryos. Moreover, to simultaneously detect protein and enable RNA probe penetration for the combined IF/smRNA FISH technique, we performed IF before smRNA FISH. We removed the zona pellucida, used Triton X-100 to permeabilize the embryos, and did not use a proteinase digestion step so as to preserve the antigens. In addition, we modified the IF technique by using RNase-free reagents to prevent RNA degradation during the IF procedure. Using this modified sequential IF/smRNA FISH technique, we have simultaneously detected protein, lncRNA, and mRNA in whole-mount preimplantation embryos. This reliable and robust protocol will contribute to the developmental biology and RNA biology fields by providing information regarding 3D expression patterns of RNA transcripts and proteins, shedding light on their biological function. |
format | Online Article Text |
id | pubmed-9577017 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-95770172022-10-19 Simultaneous visualization of RNA transcripts and proteins in whole-mount mouse preimplantation embryos using single-molecule fluorescence in situ hybridization and immunofluorescence microscopy Hazra, Rasmani Spector, David L. Front Cell Dev Biol Cell and Developmental Biology Whole-mount single-molecule RNA fluorescence in situ hybridization (smRNA FISH) in combination with immunofluorescence (IF) offers great potential to study long non-coding RNAs (lncRNAs): their subcellular localization, their interactions with proteins, and their function. Here, we describe a step-by-step, optimized, and robust protocol that allows detection of multiple RNA transcripts and protein molecules in whole-mount preimplantation mouse embryos. Moreover, to simultaneously detect protein and enable RNA probe penetration for the combined IF/smRNA FISH technique, we performed IF before smRNA FISH. We removed the zona pellucida, used Triton X-100 to permeabilize the embryos, and did not use a proteinase digestion step so as to preserve the antigens. In addition, we modified the IF technique by using RNase-free reagents to prevent RNA degradation during the IF procedure. Using this modified sequential IF/smRNA FISH technique, we have simultaneously detected protein, lncRNA, and mRNA in whole-mount preimplantation embryos. This reliable and robust protocol will contribute to the developmental biology and RNA biology fields by providing information regarding 3D expression patterns of RNA transcripts and proteins, shedding light on their biological function. Frontiers Media S.A. 2022-10-04 /pmc/articles/PMC9577017/ /pubmed/36268512 http://dx.doi.org/10.3389/fcell.2022.986261 Text en Copyright © 2022 Hazra and Spector. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Cell and Developmental Biology Hazra, Rasmani Spector, David L. Simultaneous visualization of RNA transcripts and proteins in whole-mount mouse preimplantation embryos using single-molecule fluorescence in situ hybridization and immunofluorescence microscopy |
title | Simultaneous visualization of RNA transcripts and proteins in whole-mount mouse preimplantation embryos using single-molecule fluorescence in situ hybridization and immunofluorescence microscopy |
title_full | Simultaneous visualization of RNA transcripts and proteins in whole-mount mouse preimplantation embryos using single-molecule fluorescence in situ hybridization and immunofluorescence microscopy |
title_fullStr | Simultaneous visualization of RNA transcripts and proteins in whole-mount mouse preimplantation embryos using single-molecule fluorescence in situ hybridization and immunofluorescence microscopy |
title_full_unstemmed | Simultaneous visualization of RNA transcripts and proteins in whole-mount mouse preimplantation embryos using single-molecule fluorescence in situ hybridization and immunofluorescence microscopy |
title_short | Simultaneous visualization of RNA transcripts and proteins in whole-mount mouse preimplantation embryos using single-molecule fluorescence in situ hybridization and immunofluorescence microscopy |
title_sort | simultaneous visualization of rna transcripts and proteins in whole-mount mouse preimplantation embryos using single-molecule fluorescence in situ hybridization and immunofluorescence microscopy |
topic | Cell and Developmental Biology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9577017/ https://www.ncbi.nlm.nih.gov/pubmed/36268512 http://dx.doi.org/10.3389/fcell.2022.986261 |
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