Protocol for open-source automated universal high-content multiplex fluorescence for RNA in situ analysis
Jean Descarpentrie1, Florian Bernard2, Wilfried Souleyreau3
1Department of Molecular Biology, 90187 Umeå, Sweden; Umeå Centre for Microbial Research (UCMR), Umeå University, 90187 Umeå, Sweden.
STAR Protocols
|December 7, 2024
Summary
We developed an automated protocol for analyzing RNA in cells using open-source software. This method quantifies up to 14 mRNA targets per cell, simplifying complex fluorescence in situ transcriptomics analysis.
Area of Science:
- Molecular Biology
- Bioinformatics
- Cell Biology
Background:
- In situ hybridization is crucial for visualizing RNA within cells.
- Analyzing these images for quantitative transcriptomics is often complex and labor-intensive.
Purpose of the Study:
- To present an automated, open-source protocol for high-content multiplex fluorescence in situ transcriptomics analysis.
- To simplify the quantification of multiple mRNA probes within individual cells.
Main Methods:
- Nuclei segmentation using a Fiji macro.
- Quantification of up to 14 mRNA probes per image.
- Data summarization using a custom Python application.
Main Results:
- The protocol enables automated analysis of fluorescence in situ hybridization images.
- It accurately quantifies single or co-positive cells for multiple mRNA targets.
- Results are compiled into a single spreadsheet for ease of interpretation.
Conclusions:
- This open-source protocol streamlines high-content multiplex fluorescence in situ transcriptomics.
- It provides a robust and accessible method for RNA visualization and quantification in cells.
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