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:
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.
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
Research 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 slides can also be used to acquire information such as the presence or absence of pathogens that would have been present at the time of sectioning. The DNA from these slides can be utilized to compare gene signatures of normal and diseased tissues.

Research motivation

Generally, the extraction of high-quality DNA from archived stained hematoxylin and eosin (H&E) slides is challenging. Barring commercially available expensive kits, there is a drought of reproducible methods to extract nucleic acids from histopathologically stained tissue slides. The key problem to be addressed here was coming up with new methods for DNA extraction from archived tissue slides that can be easily implemented in molecular biology labs with low resource settings worldwide.

Research objectives

The objective of the study was to standardize a protocol for DNA extraction from archived H&E tissue slides that can be further used for downstream molecular analysis from basic PCR to whole genome sequencing. Also, our objective was to come up with a method that is not only reproducible but also cost-effective.

Research 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 spectrometer and the quality was analyzed by agarose gel electrophoresis. Each sample was subjected to PCR to amplify the internal control gene GAPDH, thereby confirming the DNA intactness that could be further utilized for other downstream applications. Statistical analysis was performed to assess the different methods in terms of yield and purity of the DNA obtained.

Research 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 compared to other methods. Also, 90% of the extracted DNA showed amplifiable GAPDH gene indicating the intactness of the DNA, which in turn suggested that this DNA could be used for further molecular analysis.

Research conclusions

Our study explored the possible new methods for the extraction of PCR-friendly DNA from archived H&E-stained tissue slides for downstream molecular analysis. We tried and tested alternative methodologies for the removal of coverslip, deparaffinization of the tissues, rehydration and digestion by using simple facilities and common reagents in a basic molecular biology laboratory. We addressed the difficulties in removing the coverslip and deparaffinization of the tissues. Our data indicated that method 3 with xylene deparaffinization and 72 h digestion showed the highest yield of DNA and was also the best in terms of purity. The samples that were deparaffinized using pre-warmed xylene (about 50°C) produced greater DNA yields than concentrations of nucleic acids produced from the samples that were deparaffinized with mineral oil.

Research perspectives

In our study, we explored new possibilities of extracting DNA from archived specimens that can be used for molecular analysis. Similar to FFPE tissue blocks, quality H&E tissue slides can be critical in clinical studies and research. Since H&E slides are relatively inexpensive and easy to store, more work can be done with them. Thus recovered DNA can be utilized in the field of oncology for discriminating the mutational profile between the tumor and adjacent normal tissue or even in the field of hematology or immunology to understand the disease state, cause, and possible medication. Based on the preliminary evidence from our study, future research can focus on how to best utilize the discussed methods.