DNA Damage by Anticancer Agents and Its Repair: Mapping in Cells at the Subgene Level with Quantitative Polymerase Chain Reaction

1994 ◽  
Vol 222 (1) ◽  
pp. 236-242 ◽  
Author(s):  
K.A. Grimaldi ◽  
J.P. Bingham ◽  
R.L. Souhami ◽  
J.A. Hartley
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Damage ◽  
Anticancer Agents ◽  
Quantitative Polymerase Chain Reaction ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
In Cells

Analysis of Gene-Specific DNA Damage and Repair Using Quantitative Polymerase Chain Reaction

Methods ◽  
2000 ◽  
Vol 22 (2) ◽  
pp. 135-147 ◽  
Author(s):  
Sylvette Ayala-Torres ◽  
Yiminig Chen ◽  
Tamara Svoboda ◽  
Judah Rosenblatt ◽  
Bennett Van Houten
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Damage ◽  
Quantitative Polymerase Chain Reaction ◽  
Chain Reaction ◽  
Dna Damage And Repair ◽  
Polymerase Chain

scholarly journals Oxytetracycline induces DNA damage and epigenetic changes: a possible risk for human and animal health?

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3236 ◽  
Author(s):  
Adriana Gallo ◽  
Rosaria Landi ◽  
Valentina Rubino ◽  
Alessandro Di Cerbo ◽  
Angela Giovazzino ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Damage ◽  
Molecular Mechanisms ◽  
Mononuclear Cells ◽  
Quantitative Polymerase Chain Reaction ◽  
Animal Health ◽  
Animal Origin ◽  
Chain Reaction ◽  
Polymerase Chain

Background Oxytetracycline (OTC), which is largely employed in zootechnical and veterinary practices to ensure wellness of farmed animals, is partially absorbed within the gastrointestinal tract depositing in several tissues. Therefore, the potential OTC toxicity is relevant when considering the putative risk derived by the entry and accumulation of such drug in human and pet food chain supply. Despite scientific literature highlights several OTC-dependent toxic effects on human and animal health, the molecular mechanisms of such toxicity are still poorly understood. Methods Here, we evaluated DNA damages and epigenetic alterations by quantitative reverse transcription polymerase chain reaction, quantitative polymerase chain reaction, chromatin immuno-precipitation and Western blot analysis. Results We observed that human peripheral blood mononuclear cells (PBMCs) expressed DNA damage features (activation of ATM and p53, phosphorylation of H2AX and modifications of histone H3 methylation of lysine K4 in the chromatin) after the in vitro exposure to OTC. These changes are linked to a robust inflammatory response indicated by an increased expression of Interferon (IFN)-γ and type 1 superoxide dismutase (SOD1). Discussion Our data reveal an unexpected biological in vitro activity of OTC able to modify DNA and chromatin in cultured human PBMC. In this regard, OTC presence in foods of animal origin could represent a potential risk for both the human and animal health.

scholarly journals The Tumor-Suppressive Human Circular RNA CircITCH Sponges miR-330-5p to Ameliorate Doxorubicin-Induced Cardiotoxicity Through Upregulating SIRT6, Survivin, and SERCA2a

2020 ◽  
Vol 127 (4) ◽  
Author(s):  
Dong Han ◽  
Yongjun Wang ◽  
Yabin Wang ◽  
Xinchun Dai ◽  
Tingwen Zhou ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Polymerase Chain Reaction ◽  
Dna Damage ◽  
Quantitative Polymerase Chain Reaction ◽  
Circular Rna ◽  
Chain Reaction ◽  
Mitochondrial Oxidative Stress ◽  
Ubiquitin Protein Ligase ◽  
Polymerase Chain

Rationale: Doxorubicin is one of the most potent antitumor agents available; however, its clinical use is restricted because it poses a risk of severe cardiotoxicity. Previous work has established that CircITCH (circular RNA ITCH [E3 ubiquitin-protein ligase]) is a broad-spectrum tumor-suppressive circular RNA and that its host gene, ITCH (E3 ubiquitin protein ligase), is involved in doxorubicin-induced cardiotoxicity (DOXIC). Whether CircITCH plays a role in DOXIC remains unknown. Objective: We aimed to dissect the role of CircITCH in DOXIC and further decipher its potential mechanisms. Methods and Results: Circular RNA sequencing was performed to screen the potentially involved circRNAs in DOXI pathogenesis. Quantitative polymerase chain reaction and RNA in situ hybridization revealed that CircITCH was downregulated in doxorubicin-treated human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as well as in the autopsy specimens from cancer patients who suffered from doxorubicin-induced cardiomyopathy. Cell death/viability assays, detection of cardiomyocyte necrosis markers, microelectrode array, and cardiomyocyte functional assays revealed that CircITCH ameliorated doxorubicin-induced cardiomyocyte injury and dysfunction. Detection of cellular/mitochondrial oxidative stress and DNA damage markers verified that CircITCH alleviated cellular/mitochondrial oxidative stress and DNA damage induced by doxorubicin. RNA pull-down assays, Ago2 immunoprecipitation and double fluorescent in situ hybridization identified miR-330-5p as a direct target of CircITCH. Moreover, CircITCH was found to function by acting as an endogenous sponge that sequestered miR-330-5p. Bioinformatic analysis, luciferase reporter assays, and quantitative polymerase chain reaction showed that SIRT6 (sirtuin 6), BIRC5 (baculoviral IAP repeat containing 5, Survivin), and ATP2A2 (ATPase sarcoplasmic/endoplasmic reticulum Ca 2+ transporting 2, SERCA2a [SR Ca 2+ -ATPase 2]) were direct targets of miR-330-5p and that they were regulated by the CircITCH/miR-330-5p axis in DOXIC. Further experiments demonstrated that CircITCH-mediated alleviation of DOXIC was dependent on the interactions between miR-330-5p and the 3′-UTRs of SIRT6, BIRC5, and ATP2A2 mRNA. Finally, AAV9 (adeno-associated virus serotype 9) vector-based overexpression of the well-conserved CircITCH partly prevented DOXIC in mice. Conclusions: CircITCH represents a novel therapeutic target for DOXIC because it acts as a natural sponge of miR-330-5p, thereby upregulating SIRT6, Survivin and SERCA2a to alleviate doxorubicin-induced cardiomyocyte injury and dysfunction.

AN IMPROVED METHOD FOR INHIBITOR FREE NUCLEIC ACIDS FROM POLYPHENOL RICH LEAVES OF MUSA SPP

2017 ◽  
Vol 23 (1) ◽  
Author(s):  
N.NANDHA KUMAR ◽  
K. SOURIANATHA SUNDARAM ◽  
D. SUDHAKAR ◽  
K.K. KUMAR
Keyword(s):  
Polymerase Chain Reaction ◽  
Quantitative Polymerase Chain Reaction ◽  
Proteinase K ◽  
Chain Reaction ◽  
Banana Plant ◽  
High Molecular Weight Dna ◽  
Musa Spp ◽  
Leaf Tissues ◽  
Polymerase Chain

Excessive presence of polysaccharides, polyphenol and secondary metabolites in banana plant affects the quality of DNA and it leads to difficult in isolating good quality of DNA. An optimized modified CTAB protocol for the isolation of high quality and quantity of DNA obtained from banana leaf tissues has been developed. In this protocol a slight increased salt (NaCl) concentration (2.0M) was used in the extraction buffer. Polyvinylpyrrolidone (PVP) and Octanol were used for the removal of polyphenols and polymerase chain reaction (PCR) inhibitors. Proteins like various enzymes were degraded by Proteinase K and removed by centrifugation from plant extract during the isolation process resulting in pure genomic DNA, ready to use in downstream applications including PCR, quantitative polymerase chain reaction (qPCR), ligation, restriction and sequencing. This protocol yielded a high molecular weight DNA isolated from polyphenols rich leaves of Musa spp which was free from contamination and colour. The average yields of total DNA from leaf ranged from 917.4 to 1860.9 ng/ìL. This modified CTAB protocol reported here is less time consuming 4-5h, reproducible and can be used for a broad spectrum of plant species which have polyphenol and polysaccharide compounds.

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