Abstract 4738: Maximize recovery of quality nucleic acid from formalin-fixed paraffin-embedded tissue samples using a novel, flexible purification technology

Abstract

Abstract Formalin-fixed paraffin-embedded (FFPE) tissue samples are a valuable source of genetic information for gene expression and clinical research. Extraction of nucleic acid (NA) from FFPE tissues is a challenge because the fixation process results in cross-linking between proteins and nucleic acid, as well as between different strands of DNA or RNA molecules. Recent advances in sample preparation enables access to the valuable information contained within these difficult samples. We use a novel purification technology that does not rely on use of any harsh organic solvents and concentrates the NA for low volume elution on a specially designed spin column, thus maximizing concentration. Using this technology, we examined several aspects of the purification protocol for flexibility and adaptability: effect of de-crosslinking time on the quality and length of nucleic acids released from diverse FFPE tissues, effect of overnight storage between lysis and extraction steps, and whether the technology could be adapted to extract both DNA and RNA from the same FFPE tissue sample. Yield and quality of isolated nucleic acids were examined by absorbance, fluorescent binding dye, and real-time qPCR and RT-qPCR. We demonstrate that amplification-based quantitation gave the best indication of the quality of NA recovered. We found that a short decrosslinking time gave significant yields, but only with longer decrosslinking, times were amplifiable yields maximized for many of the tissue types tested. Preprocessing conditions were determined that allow release of both DNA and RNA for subsequent capture and elution in separate spin columns, enabling recovery of high quality DNA and RNA from the same sample in separate tubes. In this study, we demonstrate that de-crosslinking conditions can affect the recovery of amplifiable nucleic acid from FFPE samples and that RNA and DNA can be recovered from the same FFPE sample using an optimized protocol. Citation Format: Doug Wieczorek, Brad Hook, Eric Vincent, Trista Schagat. Maximize recovery of quality nucleic acid from formalin-fixed paraffin-embedded tissue samples using a novel, flexible purification technology. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4738. doi:10.1158/1538-7445.AM2015-4738