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.

Haemoprotozoan surveillance in peri-urban native and introduced wildlife from Australia

“…G2438, https://collections.qm.qld.gov.au/objects/PR121/theileria-peramelis ). While extraction of genetic material from tissue slides has been described ( Ramesh et al., 2019 ), the non-destructive sampling of blood smears from type-specimens is currently not practical. Therefore, pending further evidence, it is likely the sequences identified in the present study can be assigned to T. peramelis Mackerras, 1959 , and the host record expanded to include black rats ( R. rattus ).…”

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“…Secondly, it induces protein-nucleic acid crosslinking. Crosslinking impedes the separation of the DNA from histones, impairing the quantity as well as the quality of the extracted DNA (Ramesh et al 2019).…”

“…For the mutational analysis, DNA was isolated from slides that were prepared from FFPE blocks. A handful of studies have demonstrated the feasibility of this method as an alternative to DNA isolation directly from FFPE, which is most commonly used (Ramesh et al 2019). This approach was successful in our study.…”

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“…Subsequentemente, as amostras retiradas foram desparafinizadas por lavagens consecutivas com xileno, etanol a 100%, etanol a 70% e água destilada durante 10 minutos para cada solução. Finalmente, as amostras foram deixadas a secar durante 20 minutos à temperatura ambiente e foram colocadas em tubos PCR de 200 µl e depois para a sua conservação em congelação a -20 °C até à sua análise 10,11 (ver abaixo).…”