Imaging the Intestinal Transcriptome With Multiplexed Error-Robust Fluorescence In Situ Hybridization (MERFISH).

Area of Science:

• Molecular Biology
• Genomics
• Neuroscience

Background:

• Multiplexed Error-Robust Fluorescence In Situ Hybridization (MERFISH) is a powerful technique for spatial transcriptomic profiling.
• MERFISH has been successfully applied to brain tissues but faces challenges in other tissues due to rapid RNA degradation.

Purpose of the Study:

• To adapt and validate MERFISH protocols for challenging tissues, specifically the mammalian gastrointestinal tract.
• To provide complementary fresh-frozen and fixed-frozen protocols for MERFISH in gut tissues.
• To integrate RNA imaging with immunofluorescence for multi-modal tissue analysis.

Main Methods:

• Development of two complementary MERFISH protocols: one for fresh-frozen and one for fixed-frozen samples.
• Detailed methods for encoding probe construction, MERFISH imaging, and image decoding.
• Integration of MERFISH with immunofluorescence techniques for simultaneous RNA and protein detection.

Main Results:

• Successful application of MERFISH in mammalian gastrointestinal tract tissues, overcoming challenges of RNA degradation.
• Demonstration of combining MERFISH with immunofluorescence for comprehensive spatial profiling.
• Validation of protocols for both fresh-frozen and fixed-frozen sample preparations.

Conclusions:

• The developed MERFISH protocols are effective for spatial transcriptomic profiling in challenging tissues like the gastrointestinal tract.
• These protocols offer a valuable resource for researchers applying MERFISH to diverse and difficult-to-process tissue types.
• The integration with immunofluorescence enhances the utility of MERFISH for multi-omic spatial analysis.