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

作　　者：Pushkal Sinduvadi Ramesh Venkatesh Madegowda Suprith Kumar Shailashree Narasimha Parichay S R Nandini Nandish Manoli Devananda Devegowda 

机构地区：[1]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 [2]CIPHER Healthcare Pvt Ltd.,Hyderabad 500034,India [3]Department of Pathology,JSS Medical College,JSS Academy of Higher Education and Research,Mysuru 570015,India

出　　处：《World Journal of Methodology》2019年第3期32-43,共12页世界方法学杂志

基　　金：the junior research fellowship from the Council of Scientific and Industrial Research, Government of India

摘　　要：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.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 Nano Drop spectrophometer and the quality was analyzed by agarose gel electrophoresis. Then each sample wassubjected 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.

关 键 词：DNA extraction HEMATOXYLIN and EOSIN TISSUE SLIDES Molecular analysis POLYMERASE chain reaction Deparaffinization 

分 类 号：R73[医药卫生—肿瘤]