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 th...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.展开更多
Objective:To assess the nuclear factor-erythroid 2-related factor-2(Nrf2)modulatory effect of caffeic acid and protocatechuic acid and determine the anti-tumor activity of these phenolic compounds against Ehrlich asci...Objective:To assess the nuclear factor-erythroid 2-related factor-2(Nrf2)modulatory effect of caffeic acid and protocatechuic acid and determine the anti-tumor activity of these phenolic compounds against Ehrlich ascites carcinoma growth in mice.Methods:Antioxidant activity of protocatechuic acid and caffeic acid was assessed using ferric reducing antioxidant power(FRAP)and 2,2-diphenyl-1-picrylhydrazyl(DPPH).Nrf2 activation potential of phenolic compounds was tested by quantitative realtime polymerase chain reaction,and luciferase complementation reporter assays.In vivo efficacy was tested using the Ehrlich ascites carcinoma model.Results:FRAP and DPPH radical scavenging assays showed that caffeic acid and protocatechuic acid were more potent compared with cinnamic acid and benzoic acid.Luciferase complementation reporter assays identified caffeic acid and protocatechuic acid as the activators of Nrf2.Both caffeic acid and protocatechuic acid upregulated the expression of Nrf2 target genes heme oxygenase-1(HO-1),glutamate-cysteine ligase catalytic subunit(GCLC),and glutamate-cysteine ligase modifier subunit(GCLM)and the activity of NAD(P)H:quinone oxidoreductase 1(NQO1)when tested on HCT-116 cells using a cell-based assay system at 9 h.In addition,intraperitoneal administration of caffeic acid and protocatechuic acid to Ehrlich ascites carcinoma bearing mice suppressed tumor growth and angiogenesis.Conclusions:Caffeic acid and protocatechuic acid can modulate Nrf2 and inhibit Ehrlich ascites carcinoma cells.展开更多
基金the junior research fellowship from the Council of Scientific and Industrial Research, Government of India
文摘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.
文摘Objective:To assess the nuclear factor-erythroid 2-related factor-2(Nrf2)modulatory effect of caffeic acid and protocatechuic acid and determine the anti-tumor activity of these phenolic compounds against Ehrlich ascites carcinoma growth in mice.Methods:Antioxidant activity of protocatechuic acid and caffeic acid was assessed using ferric reducing antioxidant power(FRAP)and 2,2-diphenyl-1-picrylhydrazyl(DPPH).Nrf2 activation potential of phenolic compounds was tested by quantitative realtime polymerase chain reaction,and luciferase complementation reporter assays.In vivo efficacy was tested using the Ehrlich ascites carcinoma model.Results:FRAP and DPPH radical scavenging assays showed that caffeic acid and protocatechuic acid were more potent compared with cinnamic acid and benzoic acid.Luciferase complementation reporter assays identified caffeic acid and protocatechuic acid as the activators of Nrf2.Both caffeic acid and protocatechuic acid upregulated the expression of Nrf2 target genes heme oxygenase-1(HO-1),glutamate-cysteine ligase catalytic subunit(GCLC),and glutamate-cysteine ligase modifier subunit(GCLM)and the activity of NAD(P)H:quinone oxidoreductase 1(NQO1)when tested on HCT-116 cells using a cell-based assay system at 9 h.In addition,intraperitoneal administration of caffeic acid and protocatechuic acid to Ehrlich ascites carcinoma bearing mice suppressed tumor growth and angiogenesis.Conclusions:Caffeic acid and protocatechuic acid can modulate Nrf2 and inhibit Ehrlich ascites carcinoma cells.
