Rapid and Reliable Method To Isolate Nuclei from FFPE Specimens for Genomic-Scale Molecular Profiling.
Keith Killian, Lu Wang, Bob Walker, Art Glatfelter, William I Smith, Paul S Meltzer. National Cancer Institute, Bethesda, MD; Suburban Hospital, Bethesda, MD
Background: Surgical pathology specimens are complex mixtures of benign and malignant cell types. Germ-line and lineage-related molecular signals arising from background benign cells may mask genomic and epigenomic aberrations present in malignant cells; this effect lowers the sensitivity of somatic mutation profiling. In contrast with FISH and PCR-type assays, which can make use of a few hundred cancer-cell nuclei/genome equivalents, high-resolution genome-scale profiling requires 3 orders of magnitude more input DNA. In addition, pathology specimens are formalin-fixed and paraffin-embedded, which may present additional challenges for large-scale cell and/or organelle fractionation.
Design: This study was designed to develop a method for the large-scale, reliable and rapid purification of individual, unclumped, stable nuclei with minimal background debris from FFPE archival specimens. Details of specimen homogenization, lysis, washing, and storage were optimized to reduce processing time and improve yield using empirical measurements. Yield and purity of extracted nuclei were analyzed by DNA flow cytometry and florescence microscopy; performance in molecular profiling studies including high-resolution array comparative genomic hybridization was also tested. Technical replications were performed to test reliability of results.
Results: Details of specimen homogenization, lysis, washing, and storage were optimized to permit recovery of millions of FACS-ready nuclei from FFPE specimens in < 2 hours. High-resolution array CGH assays manifest significant improvement in the efficiency of aberration detection.
Conclusions: We present a facile and rapid method for the extraction of copious amounts of nuclei from FFPE samples, and demonstrate excellent molecular profiling results with the nuclei fractions.
Category: Special Category - Pan-genomic/Pan-proteomic approaches to Cancer
Monday, February 28, 2011 1:00 PM
Poster Session II # 242, Monday Afternoon