Quantitative Analysis of Gene Expression in RNAscope-processed Brain Tissue

Area of Science:

• Neuroscience
• Molecular Biology
• Bioinformatics

Background:

• In situ hybridization (ISH) is crucial for rodent brain molecular characterization.
• RNAscope offers sensitive, multiplexed transcript detection in single cells.
• Manual quantification of RNAscope data is time-consuming and labor-intensive.

Purpose of the Study:

• To develop a protocol for high-quality image generation for RNAscope-labeled neurons.
• To create an automated workflow for optimizing and validating cell detection parameters.
• To establish a method for deriving reliable mRNA signal thresholds.

Main Methods:

• Utilized custom scripts within the open-source software QuPath.
• Developed an automated workflow for cell detection parameter optimization.
• Implemented a method for deriving mRNA signal thresholds using negative controls.

Main Results:

• Generated a protocol for high-quality imaging of RNAscope-stained rat brain tissue.
• Created an automated QuPath workflow for reproducible cell and transcript quantification.
• Validated a method for setting accurate mRNA signal thresholds.

Conclusions:

• The protocol and automated workflow enhance the reliability and reproducibility of rodent brain tissue analysis.
• Facilitates accurate cell type characterization using RNAscope in neuroscience research.
• Aids scientists in efficiently analyzing multiplexed ISH data.