Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction

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...

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Main Authors:	Patel, Palak G., Selvarajah, Shamini, Boursalie, Suzanne, How, Nathan E., Ejdelman, Joshua, Guerard, Karl-Philippe, Bartlett, John M., Lapointe, Jacques, Park, Paul C., Okello, John B. A., Berman, David M.
Format:	Online
Language:	English
Published:	MyJove Corporation 2016
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5091935/

id	pubmed-5091935
recordtype	oai_dc
spelling	pubmed-50919352016-11-15 Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction Patel, Palak G. Selvarajah, Shamini Boursalie, Suzanne How, Nathan E. Ejdelman, Joshua Guerard, Karl-Philippe Bartlett, John M. Lapointe, Jacques Park, Paul C. Okello, John B. A. Berman, David M. Basic Protocol 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. MyJove Corporation 2016-08-21 /pmc/articles/PMC5091935/ /pubmed/27583817 http://dx.doi.org/10.3791/54299 Text en Copyright © 2016, Journal of Visualized Experiments http://creativecommons.org/licenses/by-nc-nd/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. To view a copy of this license, visithttp://creativecommons.org/licenses/by-nc-nd/3.0/
repository_type	Open Access Journal
institution_category	Foreign Institution
institution	US National Center for Biotechnology Information
building	NCBI PubMed
collection	Online Access
language	English
format	Online
author	Patel, Palak G. Selvarajah, Shamini Boursalie, Suzanne How, Nathan E. Ejdelman, Joshua Guerard, Karl-Philippe Bartlett, John M. Lapointe, Jacques Park, Paul C. Okello, John B. A. Berman, David M.
spellingShingle	Patel, Palak G. Selvarajah, Shamini Boursalie, Suzanne How, Nathan E. Ejdelman, Joshua Guerard, Karl-Philippe Bartlett, John M. Lapointe, Jacques Park, Paul C. Okello, John B. A. Berman, David M. Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction
author_facet	Patel, Palak G. Selvarajah, Shamini Boursalie, Suzanne How, Nathan E. Ejdelman, Joshua Guerard, Karl-Philippe Bartlett, John M. Lapointe, Jacques Park, Paul C. Okello, John B. A. Berman, David M.
author_sort	Patel, Palak G.
title	Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction
title_short	Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction
title_full	Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction
title_fullStr	Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction
title_full_unstemmed	Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction
title_sort	preparation of formalin-fixed paraffin-embedded tissue cores for both rna and dna extraction
description	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.
publisher	MyJove Corporation
publishDate	2016
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5091935/
_version_	1613709316979687424

Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction

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