molecular mechanisms of neurodegeneration: neurodegenerative disorders webinar series part 1
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Molecular Mechanisms of Neurodegeneration
Molecular Mechanisms of NeurodegenerationWei Cao, Ph.D.
Wei.Cao@qiagen.com
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Contact Technical Support:
BRCsupport@QIAGEN.COM1-800-362-7737
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Molecular Mechanisms of Neurodegeneration
Welcome to our three-part webinar series on neurodegeneration
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Neurodegenerative disorders: molecular mechanisms and circulating biomarker discovery – a three-part webinar series
Part 1: Molecular Mechanisms of Neurodegeneration
Part 2: The Central Roles of Non-coding RNAs in Neurodegenerative Disorders
Part 3: Circulating Biomarkers for Alzheimer’s Disease
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Molecular Mechanisms of Neurodegeneration
Legal disclaimer
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QIAGEN products shown here are intended for molecular
biology applications. These products are not intended for the
diagnosis, prevention or treatment of a disease.
For up-to-date licensing information and product-specific
disclaimers, see the respective QIAGEN kit handbook or user
manual. QIAGEN kit handbooks and user manuals are
available at www.QIAGEN.com or can be requested from
QIAGEN Technical Services or your local distributor.
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Signaling pathways in neurodegeneration4
Agenda
Molecular mechanisms of neurodegeneration1
Pathways that clean up proteins2
Aging – the greatest risk factor3
QIAGEN research solutions5
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Molecular Mechanisms of Neurodegeneration
Neurodegeneration
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Neurodegeneration is featured by progressive dysfunction and death of cells in selected areas of the nervous system
Alzheimer’s disease (AD) Parkinson’s disease (PD) Huntington’s disease (HD) Amyotrophic lateral sclerosis (ALS)
Common molecular mechanisms Abnormal protein assemblies (protein misfolding)
Late-life cell death in adulthood
Oxidative stress
Inflammation – induced neurotoxicity
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Molecular Mechanisms of Neurodegeneration
Characteristics of neurodegenerative disorders
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Lars Bertram and Rudolph E. Tanzi, “The genetic epidemiology of neurodegenerative disease”, 2005, J Clin Invest. 115(6): 1449
Selected areas of change: macroscopic and microscopic
Alzheimer’s disease
Parkinson’s disease
Amyotrophic lateral sclerosisHuntington’s disease
Lewy body dementia
Frontotemporal dementia
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Molecular Mechanisms of Neurodegeneration
Mutant genes causing neurodegeneration
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Lars Bertram and Rudolph E. Tanzi, “The genetic epidemiology of neurodegenerative disease”, 2005, J Clin Invest. 115(6): 1449
Gene mutations and accumulation of abnormal proteins and inclusion bodies are hallmarks in most neurodegenerative diseases
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Signaling pathways in neurodegeneration4
Agenda
Molecular mechanisms of neurodegeneration1
Pathways that clean up proteins2
Aging – the greatest risk factor3
QIAGEN research solutions5
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Molecular Mechanisms of Neurodegeneration
Two pathways that clean up proteins
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1. Ubiquitin – proteasome
The primary route cells use to degrade proteins
Decreased proteasome activity is observed in neurodegeneration
Mutation of a deubiquitinating enzyme (ubiquitin carboxyl-terminal hydrolase, UCHL1) has been reported in PD
Cells maintain healthy mitochondria by degrading damaged mitochondria through mitophagy; defective mitophagy is linked to PD. USP30, a deubiquitinase localized to mitochondria, antagonizes mitophagy driven by the ubiquitin ligase Parkin (Parkin ubiquitinates and tags damaged mitochondria for clearance) and protein kinase PINK1, which are encoded by two genes associated with Parkinson’s disease
2. Autophagy – lysosome
Used proteins are poor proteasome substrates
Bingol, B. et al. “The mitochondrial deubiquitinase USP30 opposes parkin-mediated mitophagy”, 2014 Nature, 510: 370
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Molecular Mechanisms of Neurodegeneration
Autophagy in neurodegenerative disorders
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Kesidou, E. et al. “Autophagy and neurodegenerative disorders”, 2013, Neural Regen Res. 2:2275
Essential for neuronal survival, autophagy removes damaged organelles and misfolded
proteins
Excessive or insufficient autophagic activity in neurons causes neurodegeneration
Appropriate autophagy (“self-eating”)
Autophagy – lysosomal activities serve the essential function in neurodegenerative diseases
Autophagy gene FunctionULK1,2 KinaseAtg2A,B Atg9/Atg2-Atg18 complexAtg4A,B,C,D E2-like enzymeAtg5 HydrolasesBeclin-1 E3-like activityAtg7 RegulatorMAP1LC3A,B,C E1-like enzymeGABARAP, L1,L2 Ubiquitin-like modifiers and regulatorsAtg9A,B Atg9/Atg2-Atg18 complexAtg10 E2-like enzymeAtg12 ModifierAtg13 RegulatorAtg14 RegulatorAtg16L1,2 E3-like activityRB1CC1 RegulatorWIPI-1 Atg9/Atg2-Atg18 complex
Human autophagy-related genes
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Molecular Mechanisms of Neurodegeneration
Apoptosis in neurodegenerative disorders
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Crosstalk between autophagy and apoptosis
Autophagy and apoptosis can be triggered by common upstream signals
Cells switch between the two responses in a mutually exclusive manner
Autophagy constitutes a stress adaptation that avoids cell death and suppresses apoptosis, however it may also exist as an alternative cell-death pathway
Autophagy and apoptosis modulation has potential as a treatment strategy in neurodegenerative diseases Caspase-dependent and caspase-
independent routes to cell death
Ghavami, S., et al. “Autophagy and apoptosis dysfunction in neurodegenerative disorders”, 2014, Progress in Neurobiology 112:24 Maiuri, M.C., et al. “Self-eating and self-killing: crosstalk between autophagy and apoptosis”, 2007, Nature Reviews, 8:741
Cell death is one of the more common mechanisms in neurodegeneration. Programmed cell death (apoptosis) regulates the number and types of cells in brain development, making it crucial for normal neural development
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Signaling pathways in neurodegeneration4
Agenda
Molecular mechanisms of neurodegeneration1
Pathways that clean up proteins2
Aging – the greatest risk factor3
QIAGEN research solutions5
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Molecular Mechanisms of Neurodegeneration
Aging – the greatest risk factor
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The greatest risk factor for neurodegeneration is aging
Genes that mediate oxidative stress responses and DNA damage repair constitute the largest classes of genes upregulated in aging
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Aging – oxidative stress and mitochondrial dysfunction
Title, Location, Date 14
Mitochondrial dysfunction is related to the aging process and neurodegenerative disorders through accumulation of mtDNA mutations and the generation of ROS
Mitochondria are highly abundant in brain cells and are both generators and targets of reactive species. Oxidative stress is inseparably linked to mitochondrial dysfunction
Mitochondrial turnover is dependent on autophagy, which declines with age and is
frequently dysfunctional in neurodegeneration. mTOR functions as an inhibitor of the
initiation step of autophagy
electron transport chains
Disease Major effective molecule Function
AD β-Amyloid Disrupts BER pathways, alters calcium homeostasis
PD Parkin Causes defective assembly of the OXPHOS complex
ALS SOD1 Binds to VDAC1, increasing oxidativedamage to mtDNA
HD Huntingtin Reduces respiratory chain complexfunction, generating ROS
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Molecular Mechanisms of Neurodegeneration
Aging – DNA damage repair
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Mutations in DNA surveillance and
DNA repair enzymes are associated
with neurodegeneration
The precise roles of DNA damage
response in the nervous system are
poorly understood
DNA damage repair is regulated
through autophagy
“The role of mitochondrial DNA mutation on neurodegenerative diseases”: http://www.ncbi.nlm.nih.gov/pubmed/25766619“The role of DNA repair in brain related disease pathology” : http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3720728/
Read review article “DNA damage repair — regulation through autophagy”https://www.qiagen.com/us/spotlight-pages/newsletters-and-magazines/articles/reviews-online-dna-damage-repair/
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Molecular Mechanisms of Neurodegeneration
PI3-K / Akt / mTOR signaling pathways in aging and neurodegeneration
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PI3-K / Akt / mTOR are therapeutic targets – may serve as essential factors for neuroprotection Cell death pathways, apoptosis and autophagy are important targets when considering
therapeutic strategies that involve PI3-K, Akt and mTOR signaling
PI3-K, Akt and mTOR pathways are tightly linked and involved in cellular senescence, aging and age-dependent diseases
mTOR is expressed at high levels in the brain
PI3-K / Akt / mTOR signaling affects a large number of cellular processes that regulate neuronal functions
Regulation of glucose metabolism Regulation of autophagy Control of mitochondrial oxidative function Modulation of lifespan – aging
Perluigi, M. et. al. “mTOR signaling in aging and neurodegeneration: At the crossroad between metabolism dysfunction and impairment of autophagy”, 2015, Neurobiology of Disease
Potential therapeutic targets
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Signaling pathways in neurodegeneration4
Agenda
Molecular mechanisms in neurodegeneration1
Pathways that clean up proteins2
Aging – the greatest risk factor3
QIAGEN research solutions5
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Molecular Mechanisms of Neurodegeneration
Download signaling pathway maps for free
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PI3k signaling
Download over 500 signaling pathway maps at QIAGEN’s website
https://www.qiagen.com/us/products/genes and pathways/pathway central/
Akt signaling mTOR signaling Apoptosis
GeneGlobe Pathway Central
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Molecular Mechanisms of Neurodegeneration
Other key signaling pathways in neurodegeneration
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Wnt
Notch
NF-κB
CREB
Wnts are constantly released in the brain to maintain basal neural activity.
"Wnts play a central role in the formation and function of neuronal circuits." (J. Mol. Cell Biol. 2014; 6: 75)
"Deregulation in Wnt signaling might have a fundamental role in the origin of neurological diseases, by altering the synaptic function at stages where the phenotype is not yet established but when the cognitive decline starts" (Front. Cell. Neurosci. 2013; 7: 1)
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Molecular Mechanisms of Neurodegeneration
Neurodegeneration is a novel class of development disorders
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Neurodegenerative diseases may represent fundamental disorders of neurogenesis and neural development
Adult neurogenesis (the emergence of new neurons in the mature brain) is altered in neurodegeneration
Signaling pathways regulate neurogenesis Wnt Notch Hedgehog Growth and neurotrophic factors BMP (bone morphogenetic proteins) Neurotransmitters Transcription factors Epigenetic modulators
Faigle R., et al. “Signaling mechanisms regulating adult neural stem cells and neurogenesis”, 2013, Biochim Biophys Acta.1830:2435
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Inflammation in neurodegeneration – neuroinflammation
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Inflammation is linked to many neurodegenerative diseases Alzheimer's disease Parkinson's disease ALS (amyotrophic lateral sclerosis) Huntington’s disease (HD)
Factors of neuroinflammation Inducers Sensors Transducers Effectors
Signaling pathways and Cytokines: TNF-α, Interleukin-1, 6 and 10, TGF-β MAPK, NF-κB, PPAR-γ
A remarkable convergence exists between the mechanisms responsible for the sensing, transduction and amplification of inflammatory responses that result in the production of neurotoxic mediators Glass, C.K., et al. “Mechanisms Underlying Inflammation in Neurodegeneration”. Cell 2010;140: 918
Molecular Mechanisms of Neurodegeneration
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microRNAs in neurodegenerative disorders
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MicroRNA (miRNA) biogenesis pathway miRNAs Highly expressed in the CNS, including the brain
and spinal cord Key modulators of both CNS development and
plasticity Have been implicated in neurodegenerative
disorders Represent a novel class of therapeutic targets for
neurodegenerative disordersDisorders Implicated miRNAs Potential Targets
AD miR-29a, miR-29b-1↓, miR-107↓, miR-298, miR-328↓ BACE1
AD miR-146a↑ complement factor H
AD miR-20a, miR-17-5p and miR-106b, miR-101 APP
AD miR-15a, miR-195 and miR-497 (only miR-15a decreased in AD) ERK1
PD miR-133b↓ PITX3
PD miR-433 FGF20
PD miR-7↓ αSYN
HD miR-9, miR-9*↓ REST, CoREST (RCOR1)
Meza-Sosa KF, et al. “Role of microRNAs in central nervous system development and pathology”, J Neurosci Res, 2012; 90:1
Molecular Mechanisms of Neurodegeneration
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Signaling pathways in neurodegeneration4
Agenda
Molecular mechanisms in neurodegeneration1
Pathways that clean up proteins2
Aging – the greatest risk factor3
QIAGEN research solutions5
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Research solutions at QIAGEN
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Visit QIAGEN’s Neurodegenerative Disease Research Portal
https://www.qiagen.com/us/products/life-science-research/neurodegenerative-disease/
Molecular Mechanisms of Neurodegeneration
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Research solutions at QIAGEN
Title, Location, Date 25
Gene expression – RT2 Profiler PCR Arrays miRNA regulation – miScript miRNA PCR Arrays DNA methylation – EpiTect DNA Methyl qPCR
Arrays DNA mutation detection – EpiTect ChIP qPCR
Arrays Copy number alteration – qBiomarker Copy
Number PCR Arrays Somatic mutation – qBiomarker Somatic Mutation
PCR Arrays ELISArray Cell-based reporter assays siRNA and shRNA mutation
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Gene expression at mRNA level – pathway-focused solutions
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Oxidative stress Unfolded protein response (ER stress) DNA damage Aging Autophagy Apoptosis Synaptic plasticity Alzheimer’s disease Parkinson’s disease Huntington’s disease Multiple sclerosis Mitochondria Neurogenesis Neuronal ion channels Neurotransmitters and receptors Neurotrophins Dopamine and serotonin pathways GABA and glutamate Pain – neuropathic and inflammatory Immunotoxicity Phagocytosis
http://www.qiagen.com/RT2-Profiler
Key signaling pathways
Cellular senescence p53 signaling PI3K-AKT signaling mTOR signaling TGFβ / BMP signaling Hedgehog signaling Notch signaling WNT signaling
RT2 Profiler PCR Array System
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Molecular Mechanisms of Neurodegeneration
RT2 Profiler PCR Array introduction
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Pathway-specific genes of
interest (84)
Housekeeping genes (5)
Genomic DNA contamination
control (GDC)
Reverse transcription
controls (RTC) (3)
Positive PCR controls (PPC)
(3)
B2M, HPRT, RPL13A, GAPDH, HGDC
Total RNA extraction cDNA mix with master-mix / load the plate run qPCR data analysis
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Molecular Mechanisms of Neurodegeneration
PCR arrays are available for 14 species!
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Human Mouse Rat Rhesus macaque Drosophila Dog Pig
Cow Chicken Horse Zebrafish Rabbit Chinese hamster ovary (CHO) C. elegans
Browse by species: https://www.qiagen.com/us/products/genes and pathways/species-portal/
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Molecular Mechanisms of Neurodegeneration
Custom PCR arrays
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RNA-seq verification Biomarker discovery CRISPR / RNAi knockdown verification Bioprocessing optimization Biological response investigation Interspecies study (14 species available)
Email: BRC.Custom@qiagen.com
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Molecular Mechanisms of Neurodegeneration
miRNA expression — miScript miRNA PCR Arrays
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miRNome Human: miRBase v21, covers 2,402 primer assays Mouse: miRBase v21, covers 1,765 primer assays Rat: 653 primer assays Dog: 277 primer assays Rhesus macaque: 469 primer assays Cow: 744 primer assays
Pathway-focused arrays (over 20 arrays) miFinder Neurological development and disease Neuropathic and inflammatory pain Apoptosis Cell development and differentiation Brain cancers Serum and plasma miRNAs
miScript PreAMP Kit Optional step for small or precious samples Full miRNome profiling from as little as 1 ng RNA
http://www.qiagen.com/products/catalog/assay-technologies/mirna/miscript-mirna-pcr-arrays
Pre-formatted, single-use PCR arrays with wet lab-verified assays
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Molecular Mechanisms of Neurodegeneration
DNA methylation screening – methylation PCR arrays
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EpiTect Methyl qPCR Array workflow
Apoptosis
Cell cycle
DNA repair
Wnt signaling
Notch signaling
Mental disorders
EpiTect DNA Methyl PCR Arrays
Analyze 22 or 94 genes per sample without bisulfite conversion
http://www.qiagen.com/products/catalog/assay-technologies/epigenetics/epitect-methyl-ii-pcr-arrays
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Molecular Mechanisms of Neurodegeneration
ChIP PCR Arrays – protein and DNA interactions
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Apoptosis
Inflammatory responses
DNA damage signaling pathway
TGFb signaling pathway
Wnt signaling pathway
Notch signaling pathway
http://www.qiagen.com/search/epitect-chip-qpcr-arrays
EpiTect ChIP qPCR Arrays
Simultaneous analysis of 84 pathway-focused genes
Requires as few as one million cells per array
Streamlined procedure, ready to use on most cyclers
Pathway-focused PCR array
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Molecular Mechanisms of Neurodegeneration
qBiomarker Copy Number PCR Arrays
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qBiomarker Copy Number PCR Arrays
Focused CNV analysis for any real-time instrument
Alzheimer’s disease
Intellectual disability
Glioma
Birth defects
Autism spectrum disorders
Schizophrenia
http://www.qiagen.com/products/catalog/assay-technologies/real-time-pcr-and-rt-pcr-reagents/qbiomarker-copy-number-pcr-arrays
qBiomarker Copy Number PCR Array layout
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Molecular Mechanisms of Neurodegeneration
Mutation detection – qBiomarker Somatic Mutation PCR Arrays
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PI3K-Akt pathway
Receptor tyrosine kinases (RTKs)
Brain cancer
http://www.qiagen.com/search/qbiomarker-somatic-mutation-pcr-arrays
Workflow
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Molecular Mechanisms of Neurodegeneration
ELISArrays – Multi-Analyte ELISArray Kits
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Analysis of multiple cytokines or chemokines
You can measure 12 cytokines or chemokines for six samples at same time!
http://www.qiagen.com/products/catalog/assay-technologies/protein-and-cell-assays/multi-analyte-elisarray-kits
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Molecular Mechanisms of Neurodegeneration
We provide service – send samples to us and receive results
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. Whole genome Illumina Gene Expression Profiling Illumina Genotyping
. Pathway-focused panel Mutation profiling Methylation PCR Array miRNA PCR array NGS
. Individual gene / locus Mutation detection Methylation qPCR NGS
. 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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Solutions at QIAGEN
Title, Location, Date 37
Gene expression RT2 Profiler PCR Arrays and Assays
Custom PCR Arrays
Epigenetics miScript miRNA PCR Arrays and Assays
EpiTect Methyl qPCR Arrays and Assays
qBiomarker Somatic Mutation PCR Arrays
qBiomarker Copy Number PCR Arrays
Protein EpiTect ChIP Arrays
ELISArray kits
Functional studies Cignal Reporter Assays
shRNA and siRNA
Service Solutions
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Thank you for attending today’s webinar!
Contact QIAGENCall: 1-800-426-8157
Email: BRCsupport@QIAGEN.com
Wei Cao, Ph.D.Wei.Cao@QIAGEN.com
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Molecular Mechanisms of Neurodegeneration
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