Proof of Concept Study: Comparison of Semi-Automated RNA Isolation Methods from Archived Formalin-Fixed, Paraffin-Embedded Tissues with Clinical Routine RNA Isolation Methods.

Abstract

High-quality RNA is crucial in clinical diagnostics and precision medicine. Formalin-fixed and paraffin-embedded (FFPE) tissues pose a challenge due to nucleic acid fragmentation and crosslinking. In this pilot study, various commercially available techniques for extracting RNA from small FFPE samples were compared. We evaluated the KingFisher Duo automated system or the manual MagMAX FFPE DNA/RNA Ultra Kit as an RNA extraction method combined with either a xylene, d-limonene, or AutoLys M tubes deparaffinization method. Additionally, the automated Maxwell RSC RNA FFPE kit and the High Pure FFPET RNA Isolation Kit were examined using FFPE samples from inflammatory bowel disease (IBD) patients, as well as samples from ovarian, kidney, and breast cancer and the skin. The KingFisher Duo system gave a higher yield and more consistent RNA quantities, especially from small volumes of IBD samples, compared to manual extraction. The deparaffinization method also impacted results, with AutoLys M tubes proving effective in combination with the KingFisher Duo system. Conversely, the High Pure kit exhibited higher yields for larger FFPE samples. While RNA integrity is a critical factor, particularly for messenger RNA (mRNA) expression studies, its role is less prominent in microRNA (miRNA) analyses. Recognizing this, our study focused on RNA yield and purity (A260/A230) to evaluate RNA extraction methods for various sample types. These findings emphasize the importance of selecting appropriate RNA extraction methods based on sample characteristics and research goals, highlighting the performance of automated methods and the impact of deparaffinization choices. The findings contribute to refining RNA extraction for molecular biology analyses, suggesting avenues for further exploration, including cost-effectiveness under specific experimental conditions.