DNA extraction from archived hematoxylin and eosin-stained tissue slides for downstream molecular analysis.

Abstract

BACKGROUNDHistopathologically 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.AIMTo standardize a new protocol for extracting DNA from archived H&E-stained tissue slides for further molecular assays.METHODSA 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.RESULTSOf 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.CONCLUSIONHere 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.