role of oxidative stress

Sample & Assay Technologies Oxidative Stress and ROS Signaling

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Overview Oxidants & antioxidant in biological

systems Antioxidant defense mechanism Molecular targets of oxidative stress Detection of oxidative stress

Research Solutions @ QIAGEN Gene expression Epigenetics Functional studies

Application Examples

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Sample & Assay Technologies Oxidative stress and ROS signaling

Oxidative stress: imbalance between reactive oxygen species (ROS) and antioxidants.

Under normal conditions, cells are able to balance the production of oxidants and antioxidants. Oxidative stress occurs when cells are subjected to excess levels of ROS or as a result of antioxidant depletion.

ROS: chemically reactive molecules containing oxygen, highly reactive

Under normal conditions, ROS are natural byproducts. It also can be generated by exogenous sources.

Major sources of ROS: mitochondria, peroxisome, plasma membrane/cytoplasm.

Role of reactive oxygen species in cell signaling pathways, Biochemical and Biomedical Aspects of Oxidative Modification 2001

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Endogenous sources

MitochondriaPeroxisomes

Cytochrome P450

Exogenous sourcesUV light

Inflammatory cytokines

Pathogens

AntioxidantsVitamin ESeleniumLycopeneGreen tea

SNPsDNA repairOxidative enzymes

ROSOxidative Stress

Damage to DNA, RNA, protein and

lipidsChromosomal

instability, gene mutations

Altered gene expression

Sample & Assay Technologies The biological importance of ROS

Physiological positive effects of ROS Kill microorganism Second messenger (H2O2) Cellular differentiation & proliferation Regulate signal transduction &

transcription

Cytokines and growth factor receptors (EGFR, PDGFR, VEGFR, receptors of TNF, IFN and IL-1)

Non-receptor tyrosine kinases (JAK, Src, MAPK and PI3K) Protein tyrosine phosphatases Serine/Threonine kinases (Akt, PKC and MAPK) Nuclear transcription factors (NF B, AP-1, NFAT, HIF-1 and

Nrf2)

Oxidative Stress and Oxidative Damage in Carcinogenesis, Toxicologic Pathology, 38: 96-109, 2010

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ROS

PLC

PI3KAkt ATM

NFkB

ERKJNKp38

NFAT

JAK

PKCp53

C-Jun

Nrf2

Gene expression changes

Sample & Assay Technologies Antioxidant defense mechanism

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Inhibit excess ROS formation, capture radicals, maintain redox homeostasis and correct mechanism of destroyed biomolecules

Mediated through Antioxidant Responsive Element (ARE) sequence Controlled by nuclear transcription factors Nrf2, AP-1, NFB, p53, NFAT

ROS Transcription factors

Transcription

ARE AREAntioxidant response elements (AREs)

Sample & Assay Technologies Nrf2-mediated oxidative stress response

Nrf2: Nuclear factor-erythroid 2-related factor is a basic leucine zipper (bZIP) transcription factor that binds to ARE to activate transcription.Keap1: Kelch-like ECH-associated protein 1. Keap1 interacts with Nrf2 in a redox-sensitive manner.

Inactive Nrf2 is retained in the cytoplasm by association with Keap1. Upon exposure to oxidative stress, Nrf2 is phosphorylated in response to the PKC, PI3K, and MAPK pathways. After phosphorylation, Nrf2 is translocated to the nucleus, binds AREs, and activates detoxifying enzymes and antioxidant enzyme.

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Sample & Assay Technologies Antioxidants maintain oxidative balance

Non-enzymatic antioxidants Alpha tocopherol (vitamin E) Ascorbic acid (vitamin C) Beta carotene

Antioxidant enzymes Superoxide dismutase (SOD) O2 + O2 + 2H+ O2 + H2O2 Catalase (CAT) 2 H2O2 2 H2O + O2 Glutathione peroxidase (GPx) 2 G-SH + R-O-OH G-SS-G + R-OH + H2O

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Sample & Assay Technologies- 9 -

Antioxidant Action of Vitamin-C

Vitamin-C is a potent antioxidant and a regulator of redox-signal transduction

Under stress, Vitamin-C acts as a primary antioxidant in plasma and cells

Vitamin-C quenches ROS and serves as a cofactor of enzymes involved in the synthesis of collagen, carnitineAnd neurotransmitters

https://www.qiagen.com/geneglobe/pathwayview.aspx?pathwayID=34&

Sample & Assay Technologies Oxidative damage of ROS

Aggregation and fragmentation

Enzyme inhibition

Proteins DNA strand breaks Mutations leading

to cancer

DNA Lipid peroxidation Damage to

membranes and lipoproteins

Membrane

ROS

Oxidative stress, disease and aging

Review article: Molecular targets of oxidative stress Biochem. J. (2011) 434, 201

What is the cellular targets of ROS that account for the toxicity?

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Sample & Assay Technologies Overview of oxidative stress & ROS

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Sample & Assay Technologies Diseases associated with oxidative stress

Diabetes

COPD

Neuro-degeneration

Asthma

Cardio-Vasculardisease

Dermatitis

Infertility

Athero-sclerosis

Cancer

OxidativeStress

inflammationChronic inflammation

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Oxidative Oxidative stress

Sample & Assay Technologies Oxidative stress links chronic inflammation & cancer

Oxidative stress, chronic inflammation, and cancer are closely linked Free radicals as a link of oxidative stress to inflammation and cancer

Oxidative stressOxidative stress

Carbonylic adducts Hydro & lipoperoxides Imbalance in DNA repair

Imbalance between pro & anti-apoptotic processes, altered gene expression & mutations

Chronic inflammation

Activation of Inflammatory cells

ROS production

LOXs, COXs, PHGPx iNOS

NFkBCytokines

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Sample & Assay Technologies ROS play multiple roles in the hallmarks of cancers

Review article: Oxidative Stress, Tumor Microenvironment, and Metabolic Reprogramming: A Diabolic Liaison International Journal of Cell Biology. 2012

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Sample & Assay Technologies

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Oxidative stress is tightly linked to mitochondrial dysfunction, and mitochondria are both generators of and targets of ROS

In respond to cellular stress, necrosis, apoptosis and autophagy are activated.

Mitochondrial turnover is dependent on autophagy

Dysregulated redox signaling or mitochondrial dysfunction affect autophagic activities

Mitochondria are the targets of ROS

Review article: Autophagy, mitochondria and oxidative stress: cross-talk and redox signaling Biochem. J. 2012, 441, 523

Sample & Assay Technologies miRNAs are modulators and targets of oxidative stress

Inflammation and cancer: interweaving microRNA, free radical, cytokine and p53 pathways Carcinogenesis, 31(1): 3749, 2010

miRNAs are non-coding RNA molecules that modulate the stability and/or the translational efficiency of target messenger RNAs

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Oxidative Stress and MicroRNAs in Vascular Diseases Int. J. Mol. Sci. 2013, 14

Relevant miRNAs in oxidative stress response

ROS source

miRNAs

upregulated Tissue/organ Source Target Functions

H2O2 miR-200c

miR-141 endothelium

miR-200a myoblasts Human ZEB1 apoptosis

miR-200b

miR-429

H2O2 miR-200c ROS accumulation;

miR-141 miR-200a ovarian Human p38 improved response to

miR-200b adenocarcinomas chemotherapy

miR-429

H2O2 miR-200c primary Mouse Unknown Unknown

hippocampal

neurons

Obesity miR-200c miR-141 Heart Rat S6K1 compensatory/adaptive

mechanisms

Sample & Assay Technologies Detection of intracellular oxidative stress

Direct approach Detection of ROS / free radicals Colorimetric (ROS-dependent reactions),

fluorometric (ROS-sensitive probes) Challenging: short-lived, highly reactive

molecules

Indirect approach Antioxidant enzyme activity Detect oxidative stress damage products Measure cellular antioxidant capacity Simple: more stable modifications of

macromolecules Wide range of techniques

Untreated Cells

Treated Cells

The cell-permeant dye H2-DCF-DA is non-fluorescent in a reduced state and exhibits fluorescence upon oxidation by ROS.

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Sample & Assay Technologies Detection of oxidative damage to macromolecules

Lipid markers of oxidative stress 4-Hydroxynonenal (HNE) is a by-product of lipid peroxidation, widely used as a

marker of chronic oxidative stress

Protein markers of oxidative stress Protein carbonylation formation of carbonyl derivatives of Pro, Lys, Arg and Thr

residues is the most common product of protein oxidation (ELISA assay). Advanced oxidation protein products (AOPP) are created when proteins react with

chlorinated oxidants.

DNA markers of oxidative stress 8-hydroxydeoxyguanosine (8-OHdG)

Antioxidant assays Catalase activity, SOD activity Total antioxidant capacity GSH levels

Avery SV. Molecular targets of oxidative stress. Biochem J. 2011 Mar 1;434(2):201-10.18


Overview Oxidants & antioxidant in biological

systems Antioxidant defense mechanism Molecular targets of oxidative stress Detection of oxidative stress

Research Solutions @ QIAGEN Gene expression Epigenetics Functional studies

Application Examples

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Sample & Assay Technologies Oxidative stress studies: experiment design

Functional studies

Epigenetics

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Gene expression

Sample & Assay Technologies Pathway-focused solution for gene expression analysis

RT2 Profiler PCR Arrays

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84 pathway-specific genes of interest

5 housekeeping genes

Genomic DNA contamination control

Reverse transcription controls (RTC) n=3

Positive PCR controls (PPC) n=3

B2M, HPRT, RPL13A, GAPDH, HGDC

Sample & Assay Technologies Application 1: gene expression

In response to cellular oxidative stress, pathways including necrosis, apoptosis, and autophagy will be activated.

.How is cell fate determined under oxidative stress?

FASEB J. 2011 October; 25(10): 3366FASEB J. 2011 October; 25(10): 3366FASEB J. 2011 October; 25(10): 3366

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Experiment design

Treat with glucocorticoidIsolate total RNA

Reverse transcription

(RT)

Data Analysis

cDNA synthesis

qPCR

Swiss-Webster mice MLO-Y4 cells

Application 1: gene expression

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1-2 pm EST, Feb. 3, 10, 17, 24, Monday, qPCR Technology webinar serieshttps://www2.gotomeeting.com/register/311390954


Pathway-focused RT2 Profiler PCR Arrays

Apoptosis RT2 Profiler PCR Array: 84 genes involved in programmed cell deathInclude: genes involved in induction of apoptosis, anti-apoptosis, regulation of apoptosis, DEATH domain proteins, caspases and regulators

Autophagy RT2 Profiler PCR Array: 84 genes involved in autophagyInclude: autophagy machinery components, regulation of autophagy

Oxidative stress RT2 Profiler PCR Array: 84 genes involved in oxidative stress


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human, mouse, rat, and up to 13 species


Oxidative Stress RT2 Profiler PCR Array- human, mouse, rat, and more

Antioxidants:Glutathione Peroxidases (GPx): GPX1, GPX2, GPX3, GPX4, GPX5, GPX6, GPX7, GSTP1, GSTZ1.

Peroxiredoxins (TPx): PRDX1, PRDX2, PRDX3, PRDX4, PRDX5, PRDX6 (AOP2).

Other Peroxidases: CAT, CYBB, CYGB, DUOX1, DUOX2, EPX, LPO, MGST3, MPO, PTGS1, PTGS2 (COX2), PXDN, TPO, TTN.

Other Antioxidants: ALB, APOE, GSR, MT3, SELS, SOD1, SOD3, SRXN1, TXNRD1, TXNRD2.

Genes Involved in ROS Metabolism:Superoxide Dismutases (SOD): SOD1, SOD2, SOD3.

Other Genes Involved in Superoxide Metabolism: ALOX12, CCS, DUOX1, DUOX2, GTF2I, MT3, NCF1, NCF2, NOS2 (iNOS), NOX4, NOX5, PREX1, UCP2.

Other Genes Involved in ROS Metabolism: AOX1, BNIP3, EPHX2, MPV17, SFTPD.

Oxidative Stress Responsive Genes: APOE, ATOX1, CAT, CCL5 (RANTES), CYGB, DHCR24, DUOX1, DUOX2, DUSP1 (PTPN16), EPX, FOXM1, FTH1, GCLC, GCLM, GPX1, GPX2, GPX3, GPX4, GPX5, GPX6, GPX7, GSR, GSS, HMOX1, HSPA1A, KRT1, LPO, MBL2, MPO, MSRA, NQO1, NUDT1, OXR1, OXSR1, PDLIM1, PNKP, PRDX2, PRDX5, PRDX6 (AOP2), PRNP, RNF7, SCARA3, SELS, SEPP1, SIRT2, SOD1, SOD2, SQSTM1, SRXN1, STK25, TPO, TTN, TXN, TXNRD1, TXNRD2.

Oxygen Transporters: CYGB, MB.


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Results: Excess GC (glucocorticoid) decreased the expression of oxidative stress responsive genes


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Sample & Assay Technologies Application 1: oxidative stress & autophagy

Results: Activation of the antioxidant genes positively correlated with activation of the autophagic

genes No significant correlation between the activation of antioxidant and antiapoptosis genes

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Sample & Assay Technologies Application 1: gene expression in oxidative stress

RT2 Profiler PCR Arrays

Apoptosis

Autophagy

Necrosis

Cell Death Pathwayfinder

Chemokines & Receptors

Cytokines & Chemokines

DNA Damage Signaling

Hypoxia Signaling

IL6/STAT3 Signalin

Inflammatory Cytokines & Receptors

Inflammatory Response & Autoimmunity

Oxidative Stress

JAK / STAT Signaling

MAP Kinase Signaling

mTOR Signaling

NFkB Signaling

TNF Signaling

Toll-Like Receptor Signaling

Transcription Factors

p53 Signaling

PI3K-AKT Signaling

VEGF Signaling

WNT Signaling

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Sample & Assay Technologies Oxidative stress: gene expression

Apoptosis:Pro-Apoptotic: ABL1, APAF1, ATP6V1G2, BAX, BCL2L11, BIRC2 (c-IAP2), CASP1 (ICE), CASP3, CASP6, CASP7, CASP9, CD40 (TNFRSF5), CD40LG (TNFSF5), CFLAR (CASPER), CYLD, DFFA, FAS (TNFRSF6), FASLG (TNFSF6), GADD45A, NOL3, SPATA2, SYCP2, TNF, TNFRSF1A, TNFRSF10A (TRAIL-R), TP53.Anti-Apoptotic: AKT1, BCL2, BCL2A1 (Bfl-1/A1), BCL2L1 (BCL-X), BIRC3 (c-IAP1), CASP2, IGF1R, MCL1, TNFRSF11B, TRAF2, XIAP.

Autophagy: AKT1, APP, ATG12, ATG16L1, ATG3, ATG5, ATG7, BAX, BCL2, BCL2L1 (BCL-X), BECN1, CASP3, CTSB, CTSS, ESR1 (ERa), FAS (TNFRSF6), GAA, HTT, IFNG, IGF1, INS, IRGM, MAP1LC3A, MAPK8 (JNK1), NFKB1, PIK3C3 (VPS34), RPS6KB1, SNCA, SQSTM1, TNF, TP53, ULK1.

Necrosis: ATP6V1G2, BMF, C1orf159, CCDC103, COMMD4, CYLD, DEFB1, DENND4A, DPYSL4, EIF5B, FOXI1, GALNT5, GRB2, HSPBAP1, JPH3, KCNIP1, MAG, OR10J3, PARP1 (ADPRT1), PARP2, PVR, RAB25, S100A7A, SPATA2, SYCP2, TMEM57, TNFRSF1A, TXNL4B.

Cell Death PathwayFinder PCR Array

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Sample & Assay Technologies Applications of oxidative stress PCR Arrays

Publications Citing the Oxidative Stress PCR Array

Susceptibility to Inhaled Flame-Generated Ultrafine Soot in Neonatal and Adult Rat Lungs, TOXICOLOGICAL SCIENCES 124(2), 472486 (2011), Jackie K. W. Chan,* Michelle V, etc.

Adaptive induction of NF-E2-related factor-2-driven antioxidant genes in endothelial cells in response to hyperglycemia, Am J Physiol Heart Circ Physiol 300: H1133H1140, 2011, Zoltan Ungvari,1 Lora Bailey-Downs, etc.

Tryptophan from human milk induces oxidative stress and upregulates the Nrf-2-mediated stress response in human intestinal cell lines. J Nutr. 2011 Aug;141(8):1417-23. Elisia I, Tsopmo A, Friel JK, etc.

Age-related gene response of human corneal endothelium to oxidative stress and DNA damage. Invest Ophthalmol Vis Sci. 2011 Mar 1;52(3):1641-9. Joyce NC, Harris DL, Zhu CC

Gene expression analysis of oxidative stress and apoptosis in proton-irradiated rat retina. In Vivo. 2010 Jul-Aug;24(4):425-30. Mao XW, Green LM, Mekonnen T, Lindsey N, Gridley DS

Increased ANG II sensitivity following recovery from acute kidney injury: role of oxidant stress in skeletal muscle resistance arteries. Am J Physiol Regul Integr Comp Physiol. 2010 Jun;298(6):R1682-91. Phillips SA, etc.

Effects of spaceflight on innate immune function and antioxidant gene expression. J Appl Physiol. 2009 Jun;106(6):1935-42. Baqai FP, Gridley DS, Slater JM, Luo-Owen X, Stodieck LS, Ferguson V, etc.

Oxidative stress: molecular perception and transduction of signals triggering antioxidant gene defenses. Braz J Med BiolRes. 2005 Jul;38(7):995-1014. Scandalios JG

Oxidative stress and gene regulation. Free Radic Biol Med. 2000 Feb 1;28(3):463-99. Allen RG, Tresini M

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Transcription factors Nrf2 and Nrf1 bind to the antioxidant response elements (ARE) and regulate genes involved in protecting cells from oxidative damages.

.How to measure the degree of antioxidant response?

Application 2: gene functions

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Sample & Assay Technologies Application 2: reporter assays for oxidative stress

Cignal Reporter Assays & Arrays

Dual-luciferase format GFP format DNA and Lenti assay 45 signaling pathways

http://www.qiagen.com/search/cignal-reporter-assay-kits32


45-pathway array & 10-Pathway ArraysSingle Pathway Assay

Antioxidant Response ReporterCignal Finder Stress & Toxicity 10-pathway reporter Kit

Measure the transcriptional activity of the Nrf2 (NF-E2-related factor 2) and Nrf1 transcription factors. Nrf2 and Nrf1 bind to the antioxidant response elements (AREs) and regulate genes involved in protecting cells from oxidative damage.

Application 2: reporter assays for oxidative stress

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Answer two questions: 1. Whether hyperglycemia activates Nrf2 in endothelial cells?2. Whether induction of Nrf2-regulated ROS detoxification systems protect

endothelial function in type 2 diabetes?

Experiment procedure: Treat wild type and Nrf2 knockout mouse with a high-fat diet (HFD) and standard diet Use reporter assay to measure Nrf2 activity


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Published data using Cignal reporter assay kit


Results & conclusion: Hyperglycemia induces oxidative stress Adaptive activation of the Nrf2/ARE pathway has a critical role in endothelial protection in response

to diabetic condition both in vitro and in vivo.


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Glucose (mmol/L)

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Functional genomics: Assess RNA interference phenotypesAssess overexpression phenotypes

Functional proteomics: Analyze the effects of recombinant protein or peptide treatments

Drug discovery: Screen and validate small molecule drug candidates

Applications: Cignal reporter assay kits

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Download a white paper Cignal Reporter Assay Kit: A high-performance tool for assessing the functions of genes, biologics, and small molecule compounds - (EN) http://www.qiagen.com/knowledge-and-support/resource-center/resource-download.aspx?id=6fc8144b-010b-43d8-9125-bf541a5d9c9c&lang=en

BenefitsExcellent sensitivity and specificityMaximal signal-to-noise ratioMinimal background transcription and rapid responseReduced experimental variation and greater reliability

Sample & Assay Technologies Application 3: antioxidant response mechanism

miRNAs play a regulatory role in Nrf2/Keap1 pathway

How is Keap1 regulated?

What is the mechanism of Nrf2-dependent antioxidant response in cancer?

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Dysregulation of the NRF2 signaling pathway is linked to cancer chemotherapy resistance

Nrf2 may contribute to tumorigenesis Nrf2 as a miRNA transcription factor A number of miRNAs are regulated by Nrf2

Sample & Assay Technologies Application 3: miRNA expression profiling

Human breast cancer cell lines MDA-MB-231 and Hs578T, non-tumorigenic MCF-10A cells

Profiled the expressions of 84 miRNAs using Human miFinder PCR Array (MIHS-001Z)

Experiment design:

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Sample & Assay Technologies miScript miRNA PCR Arrays

1 hour

2 minutes

2 hours

15 minutes

miFinder miRNA PCR Array

Profiles the expression of the 84 most abundantly expressed and best characterized miRNAs in miRBase (www.miRBase.org);

Maximize the likelihood of discovering miRNAs whose expression patterns correlate with the biological phenotypes;

Available in Human, Mouse, Rat, Dog, Rhesus Macaque, more

miRNA PCR Array miFinder Serum & Plasma Brain Cancer Breast Cancer Ovarian Cancer Prostate Cancer Cancer PathwayFinder Apoptosis Inflammatory Response and

Autoimmunity

Workflow

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Sample & Assay Technologies Application 3: miRNA expression profiling

Conclusion: miR-200a regulates the Keap1/Nrf2 pathway in mammary epithelium ; Epigenetic therapy can restore the miR-200a regulation of Keap1 expression, therefore to reactivate Nrf2-dependent antioxidant pathway in breast cancer.

Measured Nrf2 ARE promoter activity using luciferase reporter assay

Epigenetic therapy restored miR-200 expression and reduced Keap1 level

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miR-200c is a tumor suppressor miRNA, involved in cancers such as breast, ovarian, endometrial, colorectal cancer, and pancreatic cancer

Normal cell: MCF-10A

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Mature miRNAs Fold changes

has-miRNA-141 -869.07

has-miRNA-200c -422.65

has-miRNA-2302c -66.41

has-miRNA-196b -65.95

has-miRNA-143 -40.32

has-miRNA-122 -18.68

has-miRNA-7f -15.07

Sample & Assay Technologies miRNA assays and reagents from QIAGEN

Target Identification Expression

Isolation

Function

miRNA mimics & inhibitors

miScript miRNA PCR Arrays

miRNeasy

miRNAStudies

Feb 6-27, Thursday: 4 part miRNA focused webinar series, monthlyhttps://www2.gotomeeting.com/register/152689794

RT Profiler microRNA Targets PCR Arrays

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Sample & Assay Technologies Oxidative stress and DNA methylationStress & Toxicity PathwayFinder DNA Methylation PCR Array

Oxidative Stress: BNIP3, CYP1A1, GPX3, PRDX2, PTGS2 (COX2), SCARA3.

DNA Damage: ATM, BRCA1, CCND1, GADD45A, GADD45G, MLH1, MSH2, TP53, XPC.

Unfolded Protein Response: INSIG1, UBE2G2.

Hypoxia Signaling: CSTB, RARA.

Heat Shock Protein: DNAJC15.

Growth Arrest & Senescence: ATM, CDKN1A (p21CIP1/WAF1), GADD45A, GDF15 (PLAB), MSH2, TP53.

Inflammation: PRDX2, PTGS2 (COX2).

Anti-Apoptosis: BNIP3, CDKN1A (p21CIP1/WAF1), PRDX2.

Proliferation & Carcinogenesis: BRCA1, CCND1, CDKN1A (p21CIP1/WAF1), CYP1A1, PRDX2, PTGS2 (COX2), TP53.

EpiTect Methyl qPCRArray workflow

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Sample & Assay Technologies We provide service send samples to us & receive results

. Whole Genome Illumina Gene Expression Profiling Illumina Genotyping

. Pathway / Focused Panel Mutation Profiling Methylation PCR Array miRNA PCR Array

. Individual Gene / Locus Mutation Detection Methylation qPCR

. Sample Preparation DNA, RNA Extraction and Purification Cells, Tissue or Biofluids Fixed Tissue Small Sample

. http://www.qiagen.com/products/catalog/services/

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Gene Expression RT2 Profiler PCR Arrays & Assays Custom PCR Arrays

Epigenetics miScript miRNA PCR Arrays & Assays EpiTect Methyl qPCR Arrays & Assays qBiomarker Somatic Mutation PCR Arrays qBiomarker Copy Number PCR Arrays

Functional Studies Cignal Reporter Assays SureSilencing shRNA Plasmid Flexitube siRNA Flexiplate siRNA, AllStar Control

Summary

QIAGEN offers many products for oxidative stress studies

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Sample & Assay Technologies Pathway-focused solutions @ QIAGENComplete systems: sample prep through data analysis

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http://www.qiagen.com/

http://www.qiagen.com/geneglobe/

Sample & Assay Technologies- 46 -

Thank You for Attending!

Are you ready to try?Contact Technical Support

North America:

[email protected] [email protected]

Phone: 1-800-362-7737

International customers:

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Webinar related questions: [email protected]

role of oxidative stress

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