Protocol for open-source automated universal high-content multiplex fluorescence for RNA in situ analysis
Summary: In situ hybridization visualizes RNA in cells, but image analysis is complex. We present a protocol based on open-source software for automated high-content multiplex fluorescence in situ transcriptomics analysis. Steps include nuclei segmentation with a Fiji macro and quantification of up...
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| Format: | Article |
| Language: | English |
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Elsevier
2024-12-01
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| Series: | STAR Protocols |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666166724006737 |
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| author | Jean Descarpentrie Florian Bernard Wilfried Souleyreau Lucie Brisson Thomas Mathivet Ioannis S. Pateras Océane C.B. Martin Maria Lopez Chiloeches Teresa Frisan |
| author_facet | Jean Descarpentrie Florian Bernard Wilfried Souleyreau Lucie Brisson Thomas Mathivet Ioannis S. Pateras Océane C.B. Martin Maria Lopez Chiloeches Teresa Frisan |
| author_sort | Jean Descarpentrie |
| collection | DOAJ |
| description | Summary: In situ hybridization visualizes RNA in cells, but image analysis is complex. We present a protocol based on open-source software for automated high-content multiplex fluorescence in situ transcriptomics analysis. Steps include nuclei segmentation with a Fiji macro and quantification of up to 14 mRNA probes per image. We describe procedures for storing raw data, quality control images, and the use of a Python app to summarize all the results in one spreadsheet detailing the number of single or co-positive cells. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. |
| format | Article |
| id | doaj-art-4bbcbb57912d499db5fb819dc011f431 |
| institution | OA Journals |
| issn | 2666-1667 |
| language | English |
| publishDate | 2024-12-01 |
| publisher | Elsevier |
| record_format | Article |
| series | STAR Protocols |
| spelling | doaj-art-4bbcbb57912d499db5fb819dc011f4312025-08-20T02:21:07ZengElsevierSTAR Protocols2666-16672024-12-015410350810.1016/j.xpro.2024.103508Protocol for open-source automated universal high-content multiplex fluorescence for RNA in situ analysisJean Descarpentrie0Florian Bernard1Wilfried Souleyreau2Lucie Brisson3Thomas Mathivet4Ioannis S. Pateras5Océane C.B. Martin6Maria Lopez Chiloeches7Teresa Frisan8Department of Molecular Biology, 90187 Umeå, Sweden; Umeå Centre for Microbial Research (UCMR), Umeå University, 90187 Umeå, Sweden; Corresponding authorUniversity of Bordeaux, INSERM, U1212, Nucleic Acids: Natural and Artificial Regulations Laboratory, 33000 Bordeaux, FranceUniversity of Bordeaux, INSERM, U1312 BRIC, Tumor and Vascular Biology Laboratory, 33000 Bordeaux, FranceUniversity of Bordeaux, INSERM, U1312 BRIC, Tumor and Vascular Biology Laboratory, 33000 Bordeaux, FranceUniversity of Bordeaux, INSERM, U1312 BRIC, Tumor and Vascular Biology Laboratory, 33000 Bordeaux, France2nd Department of Pathology, “Attikon” University Hospital, Medical School, National and Kapodistrian University of Athens, 124 62 Athens, GreeceUniversity of Bordeaux, CNRS, IBGC, UMR 5095, 33000 Bordeaux, FranceDepartment of Molecular Biology, 90187 Umeå, Sweden; Umeå Centre for Microbial Research (UCMR), Umeå University, 90187 Umeå, SwedenDepartment of Molecular Biology, 90187 Umeå, Sweden; Umeå Centre for Microbial Research (UCMR), Umeå University, 90187 Umeå, Sweden; Corresponding authorSummary: In situ hybridization visualizes RNA in cells, but image analysis is complex. We present a protocol based on open-source software for automated high-content multiplex fluorescence in situ transcriptomics analysis. Steps include nuclei segmentation with a Fiji macro and quantification of up to 14 mRNA probes per image. We describe procedures for storing raw data, quality control images, and the use of a Python app to summarize all the results in one spreadsheet detailing the number of single or co-positive cells. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.http://www.sciencedirect.com/science/article/pii/S2666166724006737cell biologyhealth sciencesin situ hybridization |
| spellingShingle | Jean Descarpentrie Florian Bernard Wilfried Souleyreau Lucie Brisson Thomas Mathivet Ioannis S. Pateras Océane C.B. Martin Maria Lopez Chiloeches Teresa Frisan Protocol for open-source automated universal high-content multiplex fluorescence for RNA in situ analysis STAR Protocols cell biology health sciences in situ hybridization |
| title | Protocol for open-source automated universal high-content multiplex fluorescence for RNA in situ analysis |
| title_full | Protocol for open-source automated universal high-content multiplex fluorescence for RNA in situ analysis |
| title_fullStr | Protocol for open-source automated universal high-content multiplex fluorescence for RNA in situ analysis |
| title_full_unstemmed | Protocol for open-source automated universal high-content multiplex fluorescence for RNA in situ analysis |
| title_short | Protocol for open-source automated universal high-content multiplex fluorescence for RNA in situ analysis |
| title_sort | protocol for open source automated universal high content multiplex fluorescence for rna in situ analysis |
| topic | cell biology health sciences in situ hybridization |
| url | http://www.sciencedirect.com/science/article/pii/S2666166724006737 |
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