Barcoded oligonucleotides ligated on RNA amplified for multiplexed and parallel in situ analyses

Area of Science:

• Molecular Biology
• Genomics
• Neuroscience

Background:

• Accurate spatial mapping of RNA is crucial for understanding cellular function and disease.
• Existing in situ hybridization methods can be limited in multiplexing capability and require reverse transcription.

Purpose of the Study:

• To introduce barcoded oligonucleotides ligated on RNA amplified for multiplexed and parallel in situ analyses (BOLORAMIS), a reverse transcription-free method for targeted RNA identification.
• To demonstrate the capability of BOLORAMIS for single and multiplexed RNA detection in various cell types and human cerebral organoids.

Main Methods:

• BOLORAMIS utilizes ligated barcoded oligonucleotides for in situ RNA detection, avoiding the need for reverse transcription.
• The method was validated using singleplex and multiplexed experiments, including fluorescence in situ sequencing for barcode detection.
• Sensitivity and specificity were assessed by comparison with single-molecule FISH and by distinguishing between human and mouse genes.

Main Results:

• Singleplex experiments in human iPSCs revealed a stem-cell signature pattern.
• BOLORAMIS demonstrated 92% specificity, distinguishing human and mouse housekeeping genes and recapitulating lncRNA MALAT1 localization.
• Sensitivity varied for different genes (11-35%) when compared to single-molecule FISH.
• Multiplexed analysis of 96 mRNAs in a co-culture of neurons and microglial cells enabled spatial relationship and single-cell clustering analyses.

Conclusions:

• BOLORAMIS is a versatile, reverse transcription-free method for spatially-resolved, targeted in situ RNA identification.
• The technology offers high specificity and enables multiplexed analysis for uncovering spatial transcriptomic relationships.
• BOLORAMIS holds significant potential for applications in basic and translational research, particularly in neuroscience.