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Updated: Jun 24, 2026
A Simple, Quick, and Partially Automated Protocol for the Isolation of Single Nuclei from Frozen Mammalian Tissues for Single Nucleus Sequencing
Published on: July 28, 2023
Automated in situ microfluidic Random-seq for robust single-nucleus and spatial total RNA profiling of diverse FFPE
Haide Chen1,2, Yu-Sheng Chen2, Shunji Zhang1,3
1Department of Laboratory Medicine of the First Affiliated Hospital & Liangzhu Laboratory, Zhejiang University School of Medicine, Hangzhou, China.
Abstract:
Formalin-fixed paraffin-embedded (FFPE) tissues are indispensable for clinical- pathological assessment, yet widespread nucleic acid degradation severely hinders high-throughput molecular profiling. Current RNA sequencing and in situ hybridization approaches for FFPE materials suffer from inconsistent performance across tissue types and preservation conditions, restricting standardized, automated transcriptome analysis. Here, we show an in situ microfluidic Random-seq (imRandom-seq) platform that enables unified bulk, single-nucleus, and spatial total RNA profiling of FFPE specimens. Using specially designed random primers for efficient transcript capture and total transcriptome analysis, single-nucleus imRandom-seq outperforms traditional snRNA-seq and probe-based 10X Flex, with enhanced gene detection, reduced nuclear loss, and biologically reasonable cell-type annotation. Validated in difficult samples with high enzymatic activity and fragmented RNA, this in situ microfluidics-driven workflow features low manual operation, broad tissue compatibility, and robust data quality, providing a reliable and scalable tool for FFPE transcriptomic research.
Insights
A new imRandom-seq platform offers unified RNA profiling for formalin-fixed paraffin-embedded (FFPE) tissues. This method enhances gene detection and data quality for FFPE transcriptomic research.
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.
