학술논문
Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction
Document Type
research-article
Author
Palak G. Patel, author; Shamini Selvarajah, author; Suzanne Boursalie, author; Nathan E. How, author; Joshua Ejdelman, author; Karl-Philippe Guerard, author; John M. Bartlett, author; Jacques Lapointe, author; Paul C. Park, author; John B. A. Okello, author; David M. Berman, author
Source
Journal of Visualized Experiments. (114)
Subject
Language
English
ISSN
1940-087X
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.