Reproducible and sensitive micro-tissue RNA sequencing from formalin-fixed paraffin-embedded tissues for spatial gene...

Area of Science:

• Molecular Biology
• Genomics
• Pathology

Background:

• Formalin-fixed paraffin-embedded (FFPE) tissues are valuable for long-term storage and clinical research.
• However, FFPE processing can damage RNA, limiting gene expression analysis.
• Fresh-frozen tissues offer better RNA quality but lack the preservation benefits of FFPE.

Purpose of the Study:

• To develop a sensitive RNA-sequencing (RNA-seq) method for spatial transcriptome analysis of FFPE tissues.
• To assess the feasibility of analyzing gene expression in micro-dissected FFPE tissue samples.
• To evaluate the method's utility in clinical FFPE specimens, including those stored for extended periods.

Main Methods:

• Proposed a novel FFPE micro-tissue RNA-seq method.
• Combined micro-punching of tissue sections (100 μm diameter) with direct RNA-seq library construction.
• Evaluated the method on mouse liver FFPE tissues (2 years post-fixation) and clinical lung cancer FFPE specimens (6 years post-fixation).

Main Results:

• Detected approximately 7000 genes in micro-punched FFPE tissue spots (10 μm thickness).
• Achieved gene detection levels comparable to purified total RNA from equivalent cell numbers.
• Successfully identified characteristic gene expression in the tumor microenvironment of clinical FFPE lung cancer samples.

Conclusions:

• The developed FFPE micro-tissue RNA-seq method is sensitive and effective for spatial gene expression analysis.
• This technique is suitable for analyzing FFPE tissues, even those stored for many years in medical facilities.
• Enables detailed study of the tumor microenvironment in FFPE samples, supporting clinical applications.