Published online Nov 14, 2019. doi: 10.5662/wjm.v9.i3.32
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
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.
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.
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.
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.
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.
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.
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.