Automated in situ microfluidic Random-seq for robust single-nucleus and spatial total RNA profiling of diverse FFPE...

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• Formalin-fixed paraffin-embedded (FFPE) tissues are crucial for clinical pathology but suffer from nucleic acid degradation, limiting molecular profiling.
• Existing RNA sequencing and in situ hybridization methods for FFPE tissues show inconsistent performance, hindering standardized transcriptome analysis.

Purpose of the Study:

• To develop and validate an in situ microfluidic Random-seq (imRandom-seq) platform for comprehensive RNA profiling of FFPE specimens.
• To enable unified bulk, single-nucleus, and spatial total RNA analysis from FFPE tissues.

Main Methods:

• Development of an in situ microfluidic Random-seq (imRandom-seq) platform utilizing specialized random primers for efficient transcript capture.
• Application of imRandom-seq for total transcriptome analysis, including single-nucleus RNA sequencing (snRNA-seq) and spatial transcriptomics on FFPE samples.
• Validation of the platform on challenging FFPE samples with high enzymatic activity and fragmented RNA.

Main Results:

• Single-nucleus imRandom-seq demonstrated superior performance compared to traditional snRNA-seq and probe-based 10X Flex assays.
• The imRandom-seq platform achieved enhanced gene detection, reduced nuclear loss, and accurate cell-type annotation.
• The workflow showed broad tissue compatibility, low manual operation requirements, and robust data quality, even with degraded RNA.

Conclusions:

• The imRandom-seq platform provides a reliable and scalable solution for FFPE transcriptomic research.
• This in situ microfluidics-driven approach overcomes limitations of current methods, enabling standardized and automated transcriptome analysis of FFPE tissues.
• The technology facilitates advanced molecular profiling from archival FFPE samples, advancing clinical-pathological assessment and discovery.