title: Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction.
abstract: Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in molecular analysis is complicated both by heterogeneous tissue composition and low yields when extracting nucleic acids. A literature search revealed a paucity of protocols addressing these issues, and none that showed a validated method for simultaneous extraction of RNA and DNA from regions of interest in FFPET. This method addresses both issues. Tissue specificity was achieved by mapping cancer areas of interest on microscope slides and transferring annotations onto FFPET blocks. Tissue cores were harvested from areas of interest using 0.6 mm microarray punches. Nucleic acid extraction was performed using a commercial FFPET extraction system, with modifications to homogenization, deparaffinization, and Proteinase K digestion steps to improve tissue digestion and increase nucleic acid yields. The modified protocol yields sufficient quantity and quality of nucleic acids for use in a number of downstream analyses, including a multi-analyte gene expression platform, as well as reverse transcriptase coupled real time PCR analysis of mRNA expression, and methylation-specific PCR (MSP) analysis of DNA methylation.
authors: Department of Pathology & Molecular Medicine, Queen's University; Division of Cancer Biology & Genetics, Queen's Cancer Research Institute, Queen's University.
authors: Palak G
authors: PG
authors: Patel
authors: Department of Pathology & Molecular Medicine, Queen's University; Division of Cancer Biology & Genetics, Queen's Cancer Research Institute, Queen's University.
authors: Shamini
authors: S
authors: Selvarajah
authors: Department of Pathology & Molecular Medicine, Queen's University; Division of Cancer Biology & Genetics, Queen's Cancer Research Institute, Queen's University.
authors: Suzanne
authors: S
authors: Boursalie
authors: Department of Pathology & Molecular Medicine, Queen's University; Division of Cancer Biology & Genetics, Queen's Cancer Research Institute, Queen's University.
authors: Nathan E
authors: NE
authors: How
authors: Department of Surgery, Division of Urology, McGill University.
authors: Joshua
authors: J
authors: Ejdelman
authors: Department of Surgery, Division of Urology, McGill University.
authors: Karl-Philippe
authors: KP
authors: Guerard
authors: Transformative Pathology Program, Ontario Institute for Cancer Research (OICR).
authors: John M
authors: JM
authors: Bartlett
authors: Department of Surgery, Division of Urology, McGill University.
authors: Jacques
authors: J
authors: Lapointe
authors: Department of Pathology & Molecular Medicine, Queen's University.
authors: Paul C
authors: PC
authors: Park
authors: Department of Pathology & Molecular Medicine, Queen's University; Division of Cancer Biology & Genetics, Queen's Cancer Research Institute, Queen's University.
authors: John B A
authors: JB
authors: Okello
authors: Department of Pathology & Molecular Medicine, Queen's University; Division of Cancer Biology & Genetics, Queen's Cancer Research Institute, Queen's University; bermand@queensu.ca.
authors: David M
authors: DM
authors: Berman
mesh_terms: D004247:DNA / Q000302:isolation & purification*; D005557:Formaldehyde / Q000737:chemistry; D006801:Humans; D010232:Paraffin; D016612:Paraffin Embedding / Q000379:methods*; D012313:RNA / Q000302:isolation & purification*; D016707:Tissue Fixation / Q000379:methods*
chemical_list: D005557:Formaldehyde; D012313:RNA; D010232:Paraffin; D004247:DNA