Optimization of RNA Extraction from Formalin-Fixed Paraffin-Embedded Blocks for Targeted Next-Generation Sequencing

Area of Science:

• Molecular Biology
• Oncology
• Biotechnology

Background:

• Breast cancer necessitates personalized therapy, requiring molecular analysis of patient specimens.
• Fresh frozen (FF) tissues are ideal but difficult to store long-term; formalin-fixed paraffin-embedded (FFPE) tissues offer better archival stability.
• Optimizing RNA extraction from FFPE for next-generation sequencing (NGS) is crucial for advancing breast cancer research and treatment.

Purpose of the Study:

• To optimize RNA extraction from FFPE blocks for successful NGS analysis in breast cancer.
• To assess the impact of archiving time on RNA quantity and quality from FFPE samples.
• To compare the efficacy of NGS and quantitative real-time polymerase chain reaction (qRT-PCR) for RNA analysis from FFPE tissues.

Main Methods:

• RNA was extracted from paired FF and FFPE tissues (n=5).
• RNA quantity was correlated with tumor cellularity and archiving time (n=14).
• qRT-PCR and NGS were performed, with results compared across different institutions (n=96).

Main Results:

• No significant difference in RNA quality was observed between FF and FFPE specimens (p=0.49).
• Archiving time negatively impacted RNA integrity but could be overcome by experimental design, with 10-year-old samples showing 100% qRT-PCR success.
• NGS demonstrated a higher success rate than qRT-PCR; suboptimal tissue processing at one institution led to poorer outcomes.

Conclusions:

• Archived FFPE tissues are a viable source for RNA extraction for NGS if properly processed prior to fixation.
• The study provides an expected RNA yield per unit size, aiding future research.
• Proper sample handling and fixation protocols are critical for maximizing RNA quality from FFPE tissues for molecular analyses.