Multiplexed detection of RNA using MERFISH and branched DNA amplification

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• Multiplexed error-robust fluorescence in situ hybridization (MERFISH) enables spatially resolved single-cell transcriptomics by simultaneously imaging multiple RNA species.
• Limited signal brightness in MERFISH restricts applications like high-throughput imaging, short RNA detection, and analysis of challenging samples (e.g., tissues).

Purpose of the Study:

• To develop and validate a signal amplification strategy for MERFISH to overcome limitations associated with modest RNA signal brightness.
• To enhance the performance and expand the applicability of MERFISH for RNA imaging and profiling.

Main Methods:

• A branched DNA (bDNA) amplification technique was integrated with MERFISH.
• The bDNA-MERFISH approach was evaluated for signal increase, spot size, brightness variation, and detection efficiency.
• Performance was assessed using varying numbers of FISH probes per RNA species.

Main Results:

• The bDNA amplification significantly increased signal brightness in MERFISH without altering fluorescent spot size or brightness variation.
• Near 100% RNA detection efficiency was achieved with the bDNA-MERFISH method.
• Signal amplification improved MERFISH performance, particularly when using fewer probes, facilitating the imaging of shorter RNAs.

Conclusions:

• The combination of bDNA amplification and MERFISH provides a powerful tool for enhanced RNA imaging and profiling.
• This approach significantly improves MERFISH sensitivity and efficiency, broadening its utility in cell culture and tissue samples.
• The enhanced MERFISH technique is expected to facilitate a wider range of biological investigations and applications.