A44

Introduction/Background: Whether developing diagnostic tools or exploring novel therapeutic targets, cancer researchers are greatly dependent on tissue specimens that are typically available in limited quantities. Routine clinical practice worldwide includes preservation of virtually every resected biopsy and autopsy specimen. This has created valuable archived tissue collections that contain patient outcome data, such as disease progression, therapeutic response or toxicity and thus offer enormous potential for discovery and analysis of nucleic acid biomarkers with potential diagnostic and therapeutic value. However, widely used preservation methods, such as formalin-fixed paraffin-embedded (FFPE) preservation, are very harsh on a sample's RNA. The recovery of quality RNA from FFPE tissues is challenging due to the cross-linking between nucleic acids and proteins and covalent modifications of RNA that occur during the fixation process. In this study, we have evaluated the suitability of microRNAs for molecular characterization of archived human specimens and for their use as potential biomarkers.
 Materials and Methods: Total RNA was isolated from frozen and FFPE tissues using various commercial kits, and compared with respect to RNA yield, quality and recovery of microRNA. The best performing protocol was optimized and subjected to a more comprehensive characterization study, which included various tissues types, different block ages, process robustness, quantification of microRNA, rRNA and mRNA expression levels by qRT-PCR and microRNA expression profiling by microarray.
 Results: The optimized procedure, including an Armored RNA® Quant internal process control, allowed reproducible extraction of high yields of RNA from various fixed tissues. Independently of RNA sample quality, microRNAs generated a qRT-PCR signal more robust and closer to the reference frozen samples than mRNAs. In addition, the same microRNAs were found differentially expressed by qRT-PCR and microarray between two tissue types whether using FFPE or frozen samples. Although lower abundance miRNAs became undetectable upon prolonged storage, miRNA profiling allowed for classification of tissue types from specimens up to 11 years old.
 Conclusion: microRNA expression profiling and qRT-PCR are suitable platforms for retrospective molecular analysis and characterization of archived clinical samples provided that appropriate RNA isolation, quality controls and microarray methodologies are used.

Second AACR International Conference on Molecular Diagnostics in Cancer Therapeutic Development-- Sep 17-20, 2007; Atlanta, GA