Assessment of the quality of DNA from various formalin-fixed paraffin-embedded (FFPE) tissues and the use of this DNA for next-generation sequencing (NGS) with no artifactual mutation
Formalin-fixed, paraffin-embedded (FFPE) tissues used for pathological diagnosis are valuable for studying cancer genomics. In particular, laser-capture microdissection of target cells determined by histopathology combined with FFPE tissue section immunohistochemistry (IHC) enables precise analysis by next-generation sequencing (NGS) of the genetic events occurring in cancer. The result is a new strategy for a pathological tool for cancer diagnosis: ‘microgenomics’. To more conveniently and precisely perform microgenomics, we revealed by systematic analysis the following three details regarding FFPE DNA compared with paired frozen tissue DNA. 1) The best quality of FFPE DNA is obtained by tissue fixation with 10% neutral buffered formalin for 1 day and heat treatment of tissue lysates at 95°C for 30 minutes. 2) IHC staining of FFPE tissues decreases the quantity and quality of FFPE DNA to one-fourth, and antigen retrieval (at 120°C for 15 minutes, pH 6.0) is the major reason for this decrease. 3) FFPE DNA prepared as described herein is sufficient for NGS. For non-mutated tissue specimens, no artifactual mutation occurs during FFPE preparation, as shown by precise comparison of NGS of FFPE DNA and paired frozen tissue DNA followed by validation. These results demonstrate that even FFPE tissues used for routine clinical diagnosis can be utilized to obtain reliable NGS data if appropriate conditions of fixation and validation are applied.
Description
Assessment of the quality of DNA from various formalin-fixed paraffin-embedded (FFPE) tissues and the use of this DNA for next-generation sequencing (NGS) with no artifactual mutation
%0 Journal Article
%1 10.1371/journal.pone.0176280
%A Einaga, Naoki
%A Yoshida, Akio
%A Noda, Hiroko
%A Suemitsu, Masaaki
%A Nakayama, Yuki
%A Sakurada, Akihisa
%A Kawaji, Yoshiko
%A Yamaguchi, Hiromi
%A Sasaki, Yasushi
%A Tokino, Takashi
%A Esumi, Mariko
%D 2017
%I Public Library of Science
%J PLOS ONE
%K FFPE MUSTREAD benchmark dna-extraction dna-sequencing fulltext
%N 5
%P 1-18
%R 10.1371/journal.pone.0176280
%T Assessment of the quality of DNA from various formalin-fixed paraffin-embedded (FFPE) tissues and the use of this DNA for next-generation sequencing (NGS) with no artifactual mutation
%U https://doi.org/10.1371/journal.pone.0176280
%V 12
%X Formalin-fixed, paraffin-embedded (FFPE) tissues used for pathological diagnosis are valuable for studying cancer genomics. In particular, laser-capture microdissection of target cells determined by histopathology combined with FFPE tissue section immunohistochemistry (IHC) enables precise analysis by next-generation sequencing (NGS) of the genetic events occurring in cancer. The result is a new strategy for a pathological tool for cancer diagnosis: ‘microgenomics’. To more conveniently and precisely perform microgenomics, we revealed by systematic analysis the following three details regarding FFPE DNA compared with paired frozen tissue DNA. 1) The best quality of FFPE DNA is obtained by tissue fixation with 10% neutral buffered formalin for 1 day and heat treatment of tissue lysates at 95°C for 30 minutes. 2) IHC staining of FFPE tissues decreases the quantity and quality of FFPE DNA to one-fourth, and antigen retrieval (at 120°C for 15 minutes, pH 6.0) is the major reason for this decrease. 3) FFPE DNA prepared as described herein is sufficient for NGS. For non-mutated tissue specimens, no artifactual mutation occurs during FFPE preparation, as shown by precise comparison of NGS of FFPE DNA and paired frozen tissue DNA followed by validation. These results demonstrate that even FFPE tissues used for routine clinical diagnosis can be utilized to obtain reliable NGS data if appropriate conditions of fixation and validation are applied.
@article{10.1371/journal.pone.0176280,
abstract = {Formalin-fixed, paraffin-embedded (FFPE) tissues used for pathological diagnosis are valuable for studying cancer genomics. In particular, laser-capture microdissection of target cells determined by histopathology combined with FFPE tissue section immunohistochemistry (IHC) enables precise analysis by next-generation sequencing (NGS) of the genetic events occurring in cancer. The result is a new strategy for a pathological tool for cancer diagnosis: ‘microgenomics’. To more conveniently and precisely perform microgenomics, we revealed by systematic analysis the following three details regarding FFPE DNA compared with paired frozen tissue DNA. 1) The best quality of FFPE DNA is obtained by tissue fixation with 10% neutral buffered formalin for 1 day and heat treatment of tissue lysates at 95°C for 30 minutes. 2) IHC staining of FFPE tissues decreases the quantity and quality of FFPE DNA to one-fourth, and antigen retrieval (at 120°C for 15 minutes, pH 6.0) is the major reason for this decrease. 3) FFPE DNA prepared as described herein is sufficient for NGS. For non-mutated tissue specimens, no artifactual mutation occurs during FFPE preparation, as shown by precise comparison of NGS of FFPE DNA and paired frozen tissue DNA followed by validation. These results demonstrate that even FFPE tissues used for routine clinical diagnosis can be utilized to obtain reliable NGS data if appropriate conditions of fixation and validation are applied.},
added-at = {2018-01-15T20:35:29.000+0100},
author = {Einaga, Naoki and Yoshida, Akio and Noda, Hiroko and Suemitsu, Masaaki and Nakayama, Yuki and Sakurada, Akihisa and Kawaji, Yoshiko and Yamaguchi, Hiromi and Sasaki, Yasushi and Tokino, Takashi and Esumi, Mariko},
biburl = {https://www.bibsonomy.org/bibtex/25f8b1f2be7da9aa8e88696f4f0debc8c/marcsaric},
description = {Assessment of the quality of DNA from various formalin-fixed paraffin-embedded (FFPE) tissues and the use of this DNA for next-generation sequencing (NGS) with no artifactual mutation},
doi = {10.1371/journal.pone.0176280},
interhash = {f5c1baf8cb065263e8c0523a8036e3c2},
intrahash = {5f8b1f2be7da9aa8e88696f4f0debc8c},
journal = {PLOS ONE},
keywords = {FFPE MUSTREAD benchmark dna-extraction dna-sequencing fulltext},
month = {05},
number = 5,
pages = {1-18},
publisher = {Public Library of Science},
timestamp = {2018-01-15T20:35:29.000+0100},
title = {Assessment of the quality of DNA from various formalin-fixed paraffin-embedded (FFPE) tissues and the use of this DNA for next-generation sequencing (NGS) with no artifactual mutation},
url = {https://doi.org/10.1371/journal.pone.0176280},
volume = 12,
year = 2017
}