Basic Study
Copyright ©The Author(s) 2019. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Methodol. Nov 14, 2019; 9(3): 32-43
Published online Nov 14, 2019. doi: 10.5662/wjm.v9.i3.32
DNA extraction from archived hematoxylin and eosin-stained tissue slides for downstream molecular analysis
Pushkal Sinduvadi Ramesh, Venkatesh Madegowda, Suprith Kumar, Shailashree Narasimha, Parichay S R, Nandini Nandish Manoli, Devananda Devegowda
Pushkal Sinduvadi Ramesh, Venkatesh Madegowda, Suprith Kumar, Shailashree Narasimha, Devananda Devegowda, Center of Excellence in Molecular Biology and Regenerative Medicine, Department of Biochemistry, JSS Medical College, JSS Academy of Higher Education and Research, Mysuru 570015, India
Parichay S R, CIPHER Healthcare Pvt Ltd., Hyderabad 500034, India
Nandini Nandish Manoli, Department of Pathology, JSS Medical College, JSS Academy of Higher Education and Research, Mysuru 570015, India
Author contributions: Ramesh PS, S R P, and Devegowda D conceptualized the study; Ramesh PS, Madegowda V, Kumar S, and Reddy S performed all of the experiments; Devegowda D and Manoli NN aided in collecting samples and providing resources; Ramesh PS and S R P wrote the original draft; Devegowda D and Ramesh PS wrote the final draft after editing.
Institutional review board statement: The Institutional Ethical Committee of JSS Medical College and Hospital, JSS Academy of Higher Education and Research, Mysuru approved the study. All research was conducted in accordance with the approved submission.
Conflict-of-interest statement: The authors have no conflicts of interest regarding the publication of this article.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Corresponding author: Devananda Devegowda, MSc, PhD, Assistant Professor, Center of Excellence in Molecular Biology and Regenerative Medicine, Department of Biochemistry, JSS Medical College, JSS Academy of Higher Education and Research, Mysuru 570015, India. devanandd@jssuni.edu.in
Telephone: +91-961-1130458 Fax: +91-821-2548658
Received: August 5, 2019
Peer-review started: August 5, 2019
First decision: August 20, 2019
Revised: August 26, 2019
Accepted: October 15, 2019
Article in press: October 15, 2019
Published online: November 14, 2019
Processing time: 98 Days and 22.2 Hours
Abstract
BACKGROUND

Histopathologically stained archived tissue slides are stored in hospital archives for years to decades. They are the largest available source of biological materials and are a potentially useful resource that can be used for retrospective epidemiological studies. DNA recovered from the slides can be used for several downstream molecular processes including polymerase chain reaction, single nucleotide polymorphism analysis, and whole genome sequencing. The DNA from these slides can be utilized to compare gene signatures of normal and diseased tissues. However, extraction of high-quality DNA from archived stained hematoxylin and eosin (H&E) slides remains challenging.

AIM

To standardize a new protocol for extracting DNA from archived H&E-stained tissue slides for further molecular assays.

METHODS

A total of 100 archived H&E-stained cancer slides were subjected to a total of five methods of DNA extraction. Methods were varied in the deparaffinization step, tissue rehydration, duration of lysis, and presence or absence of proteinase K. The extracted DNA was quantified using a NanoDrop spectrophometer and the quality was analyzed by agarose gel electrophoresis. Then each sample was subjected to polymerase chain reaction (PCR) to amplify the internal control gene GAPDH, thereby confirming the DNA intactness, which could be further utilized for other downstream applications.

RESULTS

Of the five different methods tested, the third method wherein xylene was used for tissue deparaffinization followed by 72 h of digestion and without proteinase K inactivation yielded the highest amount of DNA with good purity. The yield was significantly higher when compared to other methods. In addition, 90% of the extracted DNA showed amplifiable GAPDH gene.

CONCLUSION

Here we present a step-by-step, cost-effective, and reproducible protocol for the extraction of PCR-friendly DNA from archived H&E-stained cancer tissue slides that can be used for further downstream molecular applications.

Keywords: DNA extraction; Hematoxylin and eosin tissue slides; Molecular analysis; Polymerase chain reaction; Deparaffinization

Core tip: In our study, we discussed a step-by-step procedure and results for the extraction of PCR-friendly DNA from archived hematoxylin and eosin-stained tissue slides. Such extracted DNA has the potential to be used for further molecular analyses such as mutation studies, whole genome sequencing, and even the identification of differences in gene signatures between diseased and normal states. Our protocol is simple, cost-effective, and can be performed in a basic molecular biology lab with most common reagents.