Optimized microfluidic spatial transcriptomic workflow for high-resolution mapping of gene expression in tissue sections

Background

Spatial transcriptomics has emerged as a crucial field in molecular biology, enabling researchers to map gene expression patterns directly within the architectural context of intact tissue sections. Unlike traditional sequencing methods that require tissue dissociation and forfeit spatial context, spatial omics preserves the physical relationships between diverse cell populations. There is a profound need for this technology to accurately decipher complex biological processes, such as embryonic development, cellular heterogeneity, and intricate cell-cell signaling networks. Understanding these spatial dynamics is essential for advancing developmental biology, identifying novel drug targets, and discovering tissue-based clinical biomarkers.

Despite its immense potential, current spatial transcriptomic approaches suffer from significant technical limitations that hinder precise biological analysis. A primary issue is suboptimal sample preparation; standard fixation methods utilizing paraformaldehyde often degrade mRNA, resulting in poor RNA preservation, truncated cDNA fragment lengths, and low overall yield. Furthermore, existing protocols struggle with low transcript capture efficiency during in situ reverse transcription. This results in inadequate detection sensitivity, typically yielding only 500 to 800 unique molecular identifiers per 100 square micrometers. Consequently, researchers face a severe trade-off between spatial resolution and data richness, preventing true single-cell resolution profiling.

Technology

This advanced spatial transcriptomics platform provides an optimized microfluidics-based workflow for mapping gene expression in tissue sections at near single-cell resolution. The solution enhances traditional deterministic barcoding through several key features. It utilizes methanol-based fixation to preserve RNA and incorporates a template-switching mechanism during in situ reverse transcription to tag intermediate cDNA sequences. Microfluidic devices then deliver two orthogonal sets of spatial barcodes through in situ ligation, creating unique identifiers for each transcript based on its exact tissue location. Finally, a second-strand synthesis step using random priming maximizes cDNA yield and quality for downstream sequencing.

What differentiates this technology is its unprecedented detection sensitivity and transcript capture efficiency compared to existing spatial omics methods. By combining methanol fixation with template switching and random priming, the platform achieves a 55% to 150% increase in unique molecular identifier (UMI) detection. It captures up to 2,000 UMIs per 100 µm² at a 10-µm resolution, which is approximately 2.5 times higher than competing methods. This superior cDNA recovery allows researchers to uncover precise biological insights, excelling in mapping complex cellular heterogeneity, discovering novel cell populations, and detailing high-resolution cell-cell interaction networks across developmental stages and disease states.

Benefits

• Significantly Enhanced Detection Sensitivity: Achieves up to a 150% increase in mRNA detection (yielding up to 2000 UMIs per 100 µm²), outperforming existing commercial methods by approximately 2.5 times.
• Near Single-Cell Spatial Resolution: Delivers highly precise spatial mapping of gene expression at a 10-µm resolution, allowing for accurate localization of transcripts within complex tissue architectures.
• Superior RNA Preservation and cDNA Yield: Utilizes methanol-based fixation, template switching during reverse transcription, and second-strand synthesis to maximize the retention, quality, and yield of mRNA.
• Advanced Biological Discovery Capabilities: Enables comprehensive mapping of cellular heterogeneity, complex cell-cell interaction networks, spatial pseudotime trajectories, and the identification of novel cell populations.
• Broad Commercial and Clinical Applicability: Highly valuable across multiple fields, including developmental biology research, pharmaceutical drug target identification, compound screening, and clinical biomarker diagnostics.
• Scalable and Multi-Omics Ready: The workflow is adaptable for robotic automation to increase throughput and consistency, and is well-positioned for future expansion into multi-omics profiling (e.g., chromatin accessibility and DNA methylation).

Applications

• Drug target identification
• Tissue-based clinical diagnostics
• Spatial biomarker discovery
• Therapeutic compound screening
• Disease mechanism research

Additional info

This microfluidics-based spatial transcriptomic platform optimizes DBiT-seq using methanol fixation, template switching during in situ reverse transcription, and random-primed second-strand synthesis. These technical refinements enhance mRNA preservation and cDNA yield, increasing unique molecular identifier detection by up to 150%. Operating at 10 µm resolution, the system provides high-sensitivity mapping of gene expression and cellular interactions in tissue sections, significantly advancing spatial omics resolution and sensitivity.