Oxidative Stress in Aging and Human Diseases - Exploring the Mechanisms

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Wei Cao, PhD
Wei.Cao@qiagen.com
Oxidative Stress in Aging and Human Diseases
– Exploring the Mechanisms
Oxidative stress and aging

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Agenda
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Oxidative stress in human diseases
Application example and solutions
Long non-coding RNAs (lncRNAs)
in oxidative stress and aging
Summary and questions

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Oxidative stress and human health
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Oxidative stress damages DNA, proteins, lipids and carbohydrates and is related to the
pathogenesis of different diseases, such as:
• Cancer
• Neurodegenerative diseases
• Cardiovascular diseases
• Obesity
• Atherosclerosis
• Diabetes
• Asthma
• Hepatic diseases
• Eye disease
• Dermatitis
• Pneumonia
• Aging
Pandey, K.B. and Rizvi, S.I. (2010) Markers of oxidative stress in erythrocytes
and plasma during aging in humans. Oxid Med Cellular Longev. 3(1), 2-12

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Reactive oxygen species (ROS) and antioxidant defense mechanisms
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• ROS have a double role: they mediate both physiologic events and cellular damage
• Antioxidants neutralize ROS/RNS by accepting or
donating electrons
◦ Endogenous enzymatic antioxidants: Superoxide
dismutase (SOD), glutathione peroxidase (GPx), catalase
(CAT) and glutathione reductase (GR)
◦ Non-enzymatic antioxidants: Vitamin C, uric acid,
albumin, bilirubin, vitamin E (α-tocopherol), β-carotene
and flavonoids
◦ Primary antioxidants: Terminate free-radical chain
reactions by donating hydrogen or electrons to free
radicals and converting them to more stable products
◦ Secondary antioxidants: Oxygen scavengers or
chelating agents
Mitochondria
Peroxisomes
Cytochrome P450
Exogenous
sources
H2O , O2 , R-OH
STABLE PRODUCTS
ANTIOXIDANT FREE RADICAL

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Markers of oxidative stress and ROS
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• The uncontrolled production of ROS/RNS plays crucial roles in
the onset of human diseases, so it is necessary to measure the
level of oxidative stress
• Oxidative stress can be quantified in vivo through the
measurement of markers which are related to ROS generation
or molecules modified by interactions with ROS
Processes Markers of oxidative stress and ROS
Lipid peroxidation
Malondialdehyde (MDA), F2-isoprostanes, oxidized low-density lipoproteins (LDL),
oxidized LDL antibodies, advanced lipid oxidation products, acrolein, 4-hydroxynonenal
Protein oxidation
Thiobarbituric acid reactive substances (TBARS), advanced oxidation protein products,
advanced glycation end-products (AGEs), disulfite formation
Carbohydrate oxidation Carbonyl formation, 3-nitrotyrosine
Nucleic acid oxidation Reactive aldehydes, reduced sugar (ascorbate, ribose, etc.), 8-oxy-2-deoxyguanosine
Serum antioxidant capacity Activity of antioxidant enzymes such as CAT, GPx, SOD
Regulators of gene expression miRNAs (such as miR-126), lncRNAs
Review article: “Today’s Oxidative Stress Biomarker” Vascular Pharmacology,
2015, 74:23

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Nrf2-ARE mediated oxidative stress response
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• Nrf2: Nuclear factor-erythroid 2-
related factor is a transcription factor
that binds to AREs (antioxidant
responsive elements) to activate
transcription
• Keap1: Kelch-like ECH-associated
protein 1
• Normal conditions: Inactive Nrf2 is
retained in the cytoplasm by
association with Keap1
• Oxidative conditions: Nrf2 is
phosphorylated, translocated to the
nucleus, where it binds AREs and
activates detoxifying enzymes and
antioxidant enzymes
• Receptor modulation: PKC, PI3K
and MAPK pathways modulate Nrf2
phosphorylation and translocation
Buendia, I. et al. (2016) Nrf2–ARE pathway: an emerging target against oxidative stress and
neuroinflammation in neurodegenerative diseases. Pharmacology & Therapeutics 157, 84–104
ARE

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Agenda
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Increased oxidative stress is linked to aging
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• Aging is characterized by a progressive loss of physiological integrity, leading to impaired
function and increased vulnerability to death
• Increased oxidative stress has been linked to aging
Lopez-Otin, C. et al. (2013) The hallmarks of aging. Cell 153, 1194-1217
Jones, D.P. (2015) Redox theory of aging. Redox Biology 5, 71-79
Giorgio, M. (2015) Oxidative stress and the unfulfilled promises of antioxidant agents. ecancer9, 556
The 9 hallmarks of aging
◦ Telomere attrition
◦ Cellular senescence
◦ Mitochondrial dysfunction
◦ Stem cell exhaustion
◦ Genomic instability
◦ Epigenetic alterations
◦ Loss of proteostasis
◦ Altered intercellular communication
◦ Deregulated nutrient sensing
◦ The redox theory of aging (also known as
the free radical theory of aging) was
proposed by Denham Harman in 1956
◦ Redox theory explains 9 hallmarks of
aging
◦ Oxidative stress increases with
increasing age. This condition leads to
accumulation of oxidation products of
lipids, nucleic acids, proteins and
carbohydrates ultimately causing cellular
dysfunction and making the body prone
to external deleterious agents
• However, several experimental models of antioxidant manipulation have failed to affect
the lifespan
• Moreover, antioxidant supplementation clinical trials have been disappointing

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Mitochondrial oxidative stress and aging
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Dai, D.F. et al. (2014) Mitochondrial oxidative stress in aging and healthspan. Longev. Healthspan 3, 6
The mitochondrial theory of aging – a revised theory
• Specifies that mitochondria are both the primary sources of ROS and the
primary targets of ROS damage; therefore, mitochondrial ROS plays a central
role in aging
• Growing experimental evidence shows the beneficial effects of mitochondrial-
targeted antioxidants in aging and healthspan
Led to a focus on developing and refining drugs to
specifically target ROS in the mitochondria of cells

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Mitochondrial oxidative stress and aging
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Dai, D.F. et al. (2014) Mitochondrial oxidative stress in aging and healthspan. Longev. Healthspan 3, 6

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Oxidative stress is linked to inflammation
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Fischer, R. and Maier, O. 2015. Interrelation of oxidative stress and inflammation
in neurodegenerative disease: role of TNF. Oxid Med Cellular Longev.
• Oxidative stress promotes inflammation
• Usually, mediators of oxidative stress and inflammation are in balance with detoxifying and
anti-inflammatory molecules
• During disease, this balance is shifted towards oxidative stress and pro-inflammatory sites,
leading to DNA, protein and cell damage, inflammation and, finally, cell death
NF𝜅B links ROS/RNS
production and the induction
of pro-inflammatory cytokines

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Aging is accompanied by inflammation
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Cannizzo, E.S. et al. 2011. Oxidative stress, inflamm-aging and immunosenescence” J Proteomics. 74(11), 2313
• Aging is accompanied by two to four-fold increases in plasma/serum levels of
inflammatory mediators such as cytokines and acute phase proteins
• Stress, declining production and function of sex hormones, genetic factors,
increased amount of fat tissue, impaired immune function, infection and
smoking/pollution all contribute to systemic chronic low-grade inflammation in
the elderly
• Aging is characterized as a systemic, chronic, low-grade inflammation. This is
called “inflamm-aging.” Inflammaging is a predictor of fragility. TNF-α and IL-6
levels are biomarkers of frailty.
• Two major pathways: oxidative stress promotes inflammatory responses:
◦ Toll like receptors (TLRs)
◦ Nalp-3 inflammasomes
Aging
Inflammation
ObservationExperiment

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NF-𝜅B pathway in aging process
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https://www.qiagen.com/us/shop/genes-and-pathways/pathway-details/?pwid=315
• NF-𝜅B is the master regulator of the
inflammatory process
• NF-𝜅B over-activation is one of the
transcriptional signatures of aging
• Mechanism of NF-𝜅B over-activation:
◦ Strongly enhanced activity of IKK and three
MAPKs (ERK, P38 and JNK) during the
aging process
◦ Increased reactive oxygen species (ROS)
production while aging
• Inhibition of NF-𝜅B prevents aging
associated features in mouse models

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Aging and the telomere connection through inflammation
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Zhang, J. et al. 2015. Ageing and the telomere connection: an intimate relationship with inflammation. Ageing Res Rev. 25, 55–69
The interplay between inflammation and telomeres/telomerase in aging process
• Mitochondria play a key role in initiating inflammatory pathways and mitochondrial dysfunction
drives the aging process
• Telomere shortening leads to accelerated ageing and increases risk of age-related diseases
• Inflammatory molecules are associated with telomere dysfunction in various diseases
• Some proteins have dual functions in both inflammation and telomere maintenance
Regulates TERT expression
NF-𝜅B
Wnt
PAPR
TERT
RAP1
Regulates TERT
expression
Involved in DNA damage
response at telomeres
Telomere lengthening and
maintenance
Shelterin complex
protein
Telomere stability
Transcription of
pro-inflammatory genes
Expressed in immune cells
Stimulate expression of
pro-inflammatory genes
Involved in NF-𝜅B
signaling pathway
Modulates NF-𝜅B
dependent transcriptions
Preserves proliferative
capacity of immune cells
In Inflammation In TelomeresProtein

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Experimental Model:
Knockout of the nfkb1 subunit of the transcription factor NF-𝜅B to induce chronic inflammation
qPCR assays to profile:
• Targets of NF-𝜅B signaling
• Genes involved oxidative stress
Application: inflammation causes aging
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Download the article: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4090717/pdf/ncomms5172.pdf
• Telomere dysfunction
• Cell senescence
• Tissue regeneration
• DNA damage
• ROS production
• COX-2 expression
nfκb1-/- mice
Aging

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Agenda
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• Experimental model: knockout of the nfkb1 subunit of the transcription factor NF-kB to
induce chronic inflammation
• qPCR arrays:
◦ NFkB Signaling Targets RT2 Profiler PCR Array
◦ Oxidative Stress RT2 Profiler PCR Array
18Oxidative stress and aging
• Telomere dysfunction
• Cell senescence
• Tissue regeneration
• DNA damage
• ROS production
• COX-2 expression
nfκb1-/- mice
Aging

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• Use QIAGEN RNeasy Mini Kit to isolate total RNA
• Use RT2 First Strand Kit for cDNA conversion
◦ Integrated DNase step
◦ Proprietary spike in RNA
◦ Priming with both oligo-dTs as well as random
hexamers
• Use RT2 SYBR Green master mix for qPCR with
RT2 Profiler PCR Arrays
◦ NFkB Signaling Targets RT2 Profiler PCR Array
◦ Oxidative Stress RT2 Profiler PCR Array
• Go to QIAGEN’s Data Analysis Center to analyze
gene expression data
Separate
beads
Experimental setup
Samples: tissues from wild type & nfκb1-/- mice
of the same age and late generation terc-/- mice

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Oxidative Stress RT2 Profiler PCR Array – 84 genes
• Human, mouse, rat, etc. up to 14 species
• 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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Oxidative Stress RT2 Profiler PCR Array

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Cytokines & chemokines:
Ccl12 (MCP-5, Scya12), Ccl22 (MDC), Ccl5 (RANTES), Csf1 (Mcsf), Csf2 (GMCSF), Csf3 (Gcsf), Cxcl1 (Gro1), Cxcl10(INP10),
Cxcl3, Cxcl9 (Mig), Fasl (Tnfsf6), Ifnb1, Ifng, Il12b, Il15, Il1a, Il1b, Il1rn, Il2, Il4, Il6, Lta (Tnfb), Ltb, Tnf, Tnfsf10(Trail)
Inflammation:
Acute Inflammation: C3, C4a, Ccl5 (RANTES), Cfb (Bf), F3, F8, Il1a, Il1b, Il6, Ins2, Stat3, Stat5b.
Other Pro-Inflammatory Genes: Agt, Akt1, Ccl12 (MCP-5, Scya12), Ccl22 (MDC), Ccr5, Cd40 (Tnfrsf5), Cxcl1 (Gro1),
Cxcl10(INP10), Cxcl3, Cxcl9 (Mig), Il15, Il1rn, Il2, Il2ra (CD25), Myd88, Ptgs2 (COX2), Sele, Selp, Tnf, Tnfrsf1b.
Apoptosis:
Agt, Birc2 (cIAP1, cIAP2), Cd74, Egfr, Fasl (Tnfsf6), Gadd45b, Ifnb1, Ifng, Il12b, Il2ra (CD25), Il4, Ins2, Lta (Tnfb), Map2k6(Mek6,
Mkk6), Mitf, Mmp9, Nqo1, Nr4a2 (Nurr1), Ptgs2 (COX2), Stat1, Tnfrsf1b, Tnfsf10 (Trail), Trp53 (p53), Traf2.
Anti-apoptotic:
Adm, Akt1, Bcl2a1a (Bfl-1, A1), Bcl2l1 (Bcl-XL), Birc3 (cIAP1, cIAP2), Ccl12 (MCP-5, Scya12), Cdkn1a (p21Cip1,
Waf1),Csf2 (GMCSF), F3, Fas (Tnfrsf6), Il1a, Il1b, Il2, Il6, Myd88, Nfkbia (Iκbα, Mad3), Sod2, Stat5b, Tnf, Xiap (Birc4).
Immune response:
Innate Immunity: C3, C4a, Ccl12 (MCP-5, Scya12), Cfb (Bf), Ifnb1, Il12b, Il6, Ins2, Myd88, Nfkbia (Iκbα, Mad3), Stat5b, Tnf.
Adaptive Immunity: C3, C4a, Ccl12 (MCP-5, Scya12), Cd40 (Tnfrsf5), Cd74, Cd80, Icam1, Ifng, Il12b, Il1b, Il2, Il4, Traf2.
Other Immune Response Genes: Cd83, Fas (Tnfrsf6), Fasl (Tnfsf6), Il1r2, Lta (Tnfb), Ltb, Tnfsf10 (Trail).
Type I interferon responsive genes:
Adm, Ccl12 (MCP-5, Scya12), Ccl5 (RANTES), Cd80, Cdkn1a (p21Cip1,
Waf1), Cfb (Bf), Cxcl10 (INP10), Cxcl9 (Mig), Il15,Il1rn, Irf1, Myd88, Ncoa3, Stat1, Tnfsf10 (Trail).
Differentiation & development:
Lymphoid Differentiation & Development: Cd80, Cd74, Cd83, Il12b, Il15, Il2, Il2ra (CD25), Il4, Irf1, Stat5b, Trp53 (p53),Vcam1.
Myeloid Differentiation & Development: Ccl5 (RANTES), Csf1 (Mcsf), Csf2 (GMCSF), Csf2rb, Csf3 (Gcsf), Il4, Mitf, Mmp9,Nfkbia (Iκbα,
Mad3), Stat5b, Tnf.
Nervous System Differentiation & Development: Agt, Aldh3a2, Cxcl1 (Gro1), Egfr, Egr2, Ifng, Nr4a2 (Nurr1), Snap25, Sod2, Stat3, Trp53
Stress responses:
Adm, Akt1, Bcl2l1 (Bcl-XL), Birc2 (cIAP1, cIAP2), Ccnd1, Cdkn1a (p21Cip1, Waf1), Gadd45b, Ifng, Il1a, Il1b, Map2k6 (Mek6,
Mkk6), Nqo1, Pdgfb, Plau (uPA), Sod2, Tnf, Trp53 (p53), Xiap (Birc4).
NFκB signaling:
Transcription Factors: Nfkb1, Nfkb2, Nfkbia (Iκbα, Mad3), Relb, Stat1.
Regulation of NFκB Signaling: Birc2 (cIAP1, cIAP2), Cd40 (Tnfrsf5), Fasl (Tnfsf6), Il1b, Myd88, Rel, Rela, Tnf, Tnfsf10(Trail).
Transcription factors:
NFκB Signaling: Nfkb1, Nfkb2, Rel, Rela, Relb.
Other Transcription Factors: Egr2, Irf1, Mitf, Myc, Nr4a2 (Nurr1), Stat1, Stat3, Stat5b, Trp53 (p53).
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NFkB Signaling Targets RT2 Profiler PCR Array

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Conclusions:
• Systemic chronic inflammation can accelerate aging via ROS-mediated exacerbation of telomere
dysfunction and cell senescence
• There exists a positive feedback loop system between telomere dysfunction, senescence-
associated ROS production and pro-inflammatory signaling that induces and stabilizes
senescence in vivo, which in turn limits the regenerative capacity of tissues

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RT2 Profiler PCR Arrays System
• Profile 84 different genes on
one array
• Appropriate controls for data
normalization, sample quality
and reaction performance
◦ Five housekeeping genes
◦ A Genomic DNA contamination
control (GDC)
◦ Three reverse transcription
controls (RTCs)
◦ Three positive PCR controls
(PPCs)
• Allows you to focus on your
questions and papers
Pathway or disease-focused gene expression profiling
Extract
total RNA
Make
cDNA
Mix with
master mix
Load plate Run qPCR
Analyze
data

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More than 170 arrays covering up to 13 species
• Aging
• DNA damage
• Oxidative stress
• Telomeres & telomerase
• Cellular senescence
• p53 signaling
• NFκB signaling
• NFκB targets
• Cell cycle
• Apoptosis
• Autophagy
• Growth factors
• mTOR signaling
• AMPK signaling
• Insulin resistance
• Stem cells
• Cancer stem cells
• Inflammatory cytokines and receptors
• Cancer inflammation and immunity
crosstalk
• Chemokines and receptors
• Common cytokines
• Cytokines and chemokines
• TNF signaling pathway
• Antiviral response
• Inflammatory response & autoimmunity
• Toll-like receptors (TLRs)
• Innate and adaptive immune responses
• Inflammasomes
• IL-6/STAT3 signaling
• T helper cell differentiation
• Th1 and Th2 responses
• Th17 response
• Interferons and receptors
• MAPK signaling
• TGFβ/BMP signaling
RT2 Profiler PCR Arrays and Assays

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Aging RT2 Profiler PCR Array
• Genomic instability: BUB1B, MRPL43, POLRMT, TFAM, TFB1M, TFB2M, ZMPSTE24
• Telomere attrition: POT1, RAP1A, TERF1, TERF2, TINF2, TPP1
• Mitochondrial dysfunction: MRPL43, NDUFB11, POLRMT, SIRT1, SIRT3, SIRT6, TFAM, TFB1M, TFB2M
• Proteostasis: ARL6IP6, BUB1B, FOXO1, HSF1, HSF1, JAKMIP3, RNF144B, SIRT1, TXNIP, VPS13C
• Laminopathies: LMNA, LMNB1, LMNB2, ZMPSTE24
• Neurodegeneration & synaptic transmission: CALB1, GFAP, MBP, SCN2B, SNAP23
• Epigenetic alterations: ARID1A, SIRT1, SIRT3, SIRT6
• DNA binding: ARID1A, ELP3, EP300, FBXL16, ZBTB10, ZFR, ZNF25
• RNA binding: ELAVL1, LSM5, ZFR
• Inflammatory response: ANGEL2, ANXA3, ANXA5, C1QA, C1QB, C1QC, C1S, C3, C3AR1, C5AR1,
CCR1, CD14, CD163, CFH, CX3CL1, CXCL16, FCER1G, FCGBP, FCGR1A, FCGR2A, FCGR3B, GFAP,
LTF, LYZ, MBP, PANX1, S100A8, S100A9, TMEM135, TMEM33, TLR2, TLR4, TOLLIP
• Apoptosis: CASP1, CLU, EP300, PDCD6, TOLLIP
• Cellular senescence: CDKN1C, VWA5A, WRN
• Cell cycle: BUB1B, CDKN1C
• Cytoskeleton: COL1A1, COL3A1, EML1
• Oxidative stress: EP300, GSTA1
• Transcriptional regulation: ARID1A, EP300, FOXO1, HSF1, PHF3, SMAD2
Oxidative stress and aging 25

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Agenda
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Long non-coding RNAs (lncRNAs): new players in aging
Grammatikakis, I. et al. (2014) Long noncoding RNAs (lncRNAs) and the molecular hallmarks of aging. Aging 6(12), 992-1009
The roles of lncRNAs in aging:
• Modulate telomere length
• Control epigenetic alterations in
aging and senescence
• Associated with proteostasis,
including autophagy and protein
synthesis, trafficking, assembly
and degradation
• Modulate stem cell homeostasis
• Involved in cell cycle regulation
• Regulate intercellular
communication
Oxidative stress and aging 27

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• lncRNAs are non-protein-coding transcripts longer than 200
nucleotides
• Most lncRNAs are localized in the nucleus, but some are found
in the cytoplasm
• Many lncRNAs are molecularly indistinguishable from mRNAs
and share many features of mRNAs
• Although some lncRNAs (e.g. MALAT1) are highly abundant
transcripts, many lncRNAs are less so. But low transcription
levels do not necessarily reflect lack of functionality
• lncRNAs may or may not contain a poly-A tail (mRNAs have a
poly-A tail)
• lncRNAs are typically less conserved across species and often
show low expression levels and high tissue specificity
• Expression of lncRNAs is generally lower than that of mRNAs
and sensitive method such as qPCR are needed to detect and
quantify them
Schwarzenbach, H. et. al. (2013) Cell-free nucleic acids as biomarkers in cancer patients. Nat. Rev. Cancer 11, 426
Rönnau, C.G.H. (2014) Noncoding RNAs as novel biomarkers in prostate cancer. Biomed. Res. Int. 591703
What are lncRNAs?

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lncRNA classification and subgroup
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Wu P. et al. (2013) Roles of long noncoding RNAs in brain development, functional
diversification and neurodegenerative diseases. Brain Research Bulletin 97, 69
• lncRNAs can exceed 100,000 nucleotides in length and
cover a wide range of gene positions
• lncRNAs can be divided into three general categories:
◦ Transcribed relative to host protein-coding genes
◦ Transcribed from gene regulatory regions
◦ Transcribed from specific chromosomal regions
Intergenic
Intronic
Exonic
Overlapping
Sense
Antisense
Classifying lncRNAs based on their relative position to
PCG (protein-coding genes)

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The diverse functions of lncRNAs

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lncRNAs in oxidative stress and aging-related disorders
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• Under oxidative stress
◦ Expression of lncRNAs change during oxidative stress
◦ lncRNAs are induced during various types of stress, including genotoxic stress, oxidative
stress and endoplasmic reticulum (ER) stress
◦ They are involved in the heat shock response, DNA damage response and hypoxia
• Aging process
◦ lncRNAs are deregulated in aging-related diseases and serve as potential molecular markers
and therapeutic targets

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Application: oxidative stress-induced changes in lncRNAs
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Study: The effect of oxidative stress on
the transcriptome of human fibroblasts
Methods:
• Cell culture: MRC5 and BJ fibroblasts
• Oxidative conditions: treated cells for 30
minutes or 2 hours with 0.2 mM H2O2
• Isolated total RNA from treated cells
Findings:
• Detected that 14,639 (68.7%) out of 21,311 lncRNAs were up-regulated
• Four lncRNAs groups are prominent:
◦ dncRNAs (65.1%)
◦ antisense overlapping lncRNAs (65.9%);
◦ terminal-associated lncRNAs transcribed at the same direction as their protein-coding genes pairs (70.7%)
◦ promoter-associated antisense lncRNAs (75.9%)
Stress-induced lncRNAs are an integral part of the core transcriptional response to
environmental stress and may facilitate cellular adaptation to stress
Analysis:
• Used RNA-seq, ChIP-seq and microarrays to analyze the genomic response: upstream, coding and
downstream
• RT-qPCR used to verify results

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Current lncRNA quantification approaches
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• RNA-seq (whole transcriptome sequencing): discover new RNAs and splicing variants
• Microarrays: use data analysis approaches to identify lncRNAs
• Real-time PCR based approaches: sensitive and quantitative for low-expressing
RNAs and small gene changes; gold standard for gene quantification; able to use
pre-amplification and WTA strategies (FFPE and single-cell analysis)
Test and verify your hypothesis with: RT2 lncRNA qPCR Assays or Custom PCR Arrays
miRNeasy and
exoRNeasy
Serum/Plasma Kit
RT² lncRNA
PreAMP PCR Kit
RT2 lncRNA
PCR System
Free data
analysis tool
Sample
isolation
Amplification qPCR
Data analysis
& interpretation

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RT2 lncRNA qPCR system
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• lncRNA databases: In-house database at QIAGEN GeneGlobe,
currently covering human GENCODE19, mouse GENCODE M2,
RefSeq Release 65 and more than 40,000 human and 27,000 mouse
lncRNA assays
• RT2 lncRNA assays: Laboratory-verified for optimal qPCR
performance with high specificity, amplification efficiency and
sensitivity
• RT2 lncRNA qPCR Arrays: Pathway or disease relevant lncRNA
assays to facilitate comparative discovery in cancer and other
research fields
◦ RT2 lncFinder PCR Array (human and mouse)
◦ RT2 lncRNA Cancer PathwayFinder Array (human and mouse)
◦ RT2 lncRNA Inflammatory Response & Autoimmunity (human and mouse)
◦ RT2 lncRNA Cell Development & Differentiation (human and mouse)
• Custom option: Flexible custom design from the lncRNA and qPCR
databases allows profiling of mRNAs and lncRNAs simultanously
• Data analysis: Free on-line data analysis tools
https://www.qiagen.com/us/search/rt2-lncrna-pcr-arrays/

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RT2 lncRNA qPCR Array – format and controls
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• Flexible layout and patented controls
• Each 96-well plate has:
◦ 84 lncRNA-specific assays
◦ Five reference genes (3 mRNAs and 2 lncRNAs)
◦ A genomic DNA control (GDC)
◦ Three reverse transcription control (RTC)
◦ Three PCR controls (PPC)
384-well format
(4x96)
RT2 Custom Array Builder: easy and flexible
96-well format
100-well ring

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Build your custom PCR array at:
https://www.qiagen.com/myPCRarray
Integrate coding & noncoding RNA expression analysis
How to get started with the new custom array builder? Watch a movie:
https://attendee.gotowebinar.com/recording/6162050561736908290
Gene expression regulates biology
Integrate coding and noncoding
RNA expression analysis
Select mRNA and
lncRNA assays to build
your custom PCR array

Sample to Insight
. Whole genome
• Illumina gene expression profiling
• Illumina genotyping
. Pathway/focused panel
• Mutation profiling
• Methylation
• PCR arrays
• miRNA PCR arrays
• NGS
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. Individual gene/locus
• Mutation detection
• Methylation
• qPCR
. Sample preparation – DNA, RNA
extraction and purification
• Cells, tissue or biofluids
• Fixed tissue
• Small samples
Visit service: http://www.qiagen.com/products/catalog/services/
Contact: BRC.Service@qiagen.com
We provide service – send your samples to us and receive results

Sample to Insight
Agenda
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5
1
3
4
2

Sample to Insight
Study oxidative stress and aging
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• Cellular pathways require molecular signaling for their activity
◦ RT2 Profiler PCR Arrays
◦ RT2 lncRNA PCR Arrays
◦ RT2 Custom PCR Arrays: interaction of coding and non-coding RNAs
• QIAGEN offers Sample to Insight solutions
◦ Sample prep
◦ Real-time PCR assays
◦ Data analysis & interpretation
• Service Core
◦ Send us your samples and receive results
Choose QIAGEN and turn your hypotheses into actionable insights!

Sample to Insight
Thank you for attending
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Thank you for attending today’s webinar!
Contact QIAGEN
Call: 1-800-426-8157
Email: BRCsupport@QIAGEN.com
QIAwebinars@QIAGEN.com
Wei Cao, PhD
Wei.Cao@QIAGEN.com
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Oxidative Stress in Aging and Human Diseases - Exploring the Mechanisms

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