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Sample & Assay Technologies
Signal transduction targets: Research solutions for pathway analysisJennifer Gibbons, Ph.D.R&D Scientist
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Sample & Assay Technologies Biology-focused solutions for pathway analysis
Signal transduction targets, Frederick, MD, 3/15/13 2
• SABiosciences is now a Company
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Sample & Assay Technologies Background: Signal transduction
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The process of disseminating an extracellular signal, resulting in a functional response
Ligand ReceptorDownstream
Signaling
Transcription Factor
Target Gene Expression
Biological Function
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Sample & Assay Technologies Background: Signal transduction
Developmental-related pathways� TGFβ superfamily� WNT� Notch� Hedgehog
Immune/Inflammatory response pathways� NFκB� JAK/STAT
Stress-related pathways� p53� Oxidative Stress� Hypoxia
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Sample & Assay Technologies TGFβ superfamily
Four signaling pathways� BMP (Bone Morphogenic Protein)
� Development/differentiation (not just bone)– Stem Cells– CNS
� TGFβ (Transforming Growth Factor Beta)� Proliferation� Differentiation
� Activin� Growth & development� Reproduction
� Nodal� Development
Pathological dysregulation� Fibrosis� Cancer
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Sample & Assay Technologies TGFβ superfamily signaling pathways
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http://www.genome.jp/kegg/pathway/hsa/hsa04350.html
BMP
TGFβ
Activin
Nodal
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Sample & Assay Technologies TGFβ superfamily signaling pathways
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http://www.genome.jp/kegg/pathway/hsa/hsa04350.html
Ligands Receptors Signaling/TFs Target Genes
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Sample & Assay Technologies WNT signaling
Three signaling pathways� Canonical (β-catenin signaling)
� Development� Cell cycle/proliferation
� Planar cell polarity� Cellular polarization� Cytoskeletal changes
� Calcium� Cell adhesion
Pathological dysregulation� Cancer� Neurodegenerative diseases� Pulmonary fibrosis
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Sample & Assay Technologies WNT signaling pathways
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http://www.genome.jp/kegg/pathway/hsa/hsa04310.html
Canonical
PCP
WNT/Ca2+
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Sample & Assay Technologies WNT signaling pathways
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http://www.genome.jp/kegg/pathway/hsa/hsa04310.html
TFsTarget Genes
DownstreamSignalingReceptorsLigands
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Sample & Assay Technologies Other signaling pathways
Notch signaling� Biological processes
� Development & differentiation� Molecular mechanism
� Receptor processing– γ-secretase complex
p53 signaling� Biological processes
� DNA damage� Oncogenesis
Molecular mechanism� Transcription factor (p53) regulation
JAK/STAT signaling� Biological processes
� Immune & inflammatory response� Molecular mechanism
� JAK activation of STATs by phosphorylation� STAT homo/heterodimerization for gene transcription
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Sample & Assay Technologies Notch signaling pathway
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http://www.genome.jp/kegg/pathway/hsa/hsa04330.html
Ligands/receptors/cofactors TFsTargetgenes
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Sample & Assay Technologies p53 signaling pathway
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http://www.genome.jp/kegg/pathway/hsa/hsa04115.html
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Sample & Assay Technologies JAK/STAT signaling pathway
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Sample & Assay Technologies Signal transduction experimental design
Gene expression� RT-PCR
Epigenetics� miRNA� DNA methylation� Histone modifications
Functional studies� Transcription factor reporter assays� siRNA/shRNA
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QIAGEN offers a variety of products for signal transduction
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Sample & Assay Technologies Principles of qRT-PCR: Overview
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Ct values: Threshold cycle
Real-time PCR� Amplify and simultaneously quantify target DNAReverse transcription real-time PCR� Amplify and simultaneously quantify mRNA
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Sample & Assay Technologies Gene expression: Which pathway to choose?
Hypothesis: An ω-3 diet (unlike the ω-6 Western diet) will suppress prostate tumorigenesis
Methods: After confirming phenotypic effect, identify the molecular mechanism using PAMM-014 (RT2 Profiler Signal Transduction PathwayFinder)
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Focus your research with the RT2 Profiler Signal Transduction PathwayFinder
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Sample & Assay Technologies Mouse Signal Transduction PathwayFinder
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Functional gene groupings identify what pathways may be affected in your experimental system
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Sample & Assay Technologies Western blots confirm PCR array results
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Sample & Assay Technologies Maybe WNT signaling involved?
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Sample & Assay Technologies Gene expression: Which pathway to choose?
Hypothesis: An ω-3 diet (unlike the ω-6 Western diet) will suppress prostate tumorigenesis
Methods: After confirming phenotypic effect, identify the molecular mechanism using PAMM-014 (RT2 Profiler Signal Transduction PathwayFinder)
Conclusions: NFκB signaling is increased during prostate cancer tumorigenesis, whereas apoptosis is decreased. These effects are reversed during the ω-3 diet, inhibiting tumorigenesis.
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Focus your research with the RT2 Profiler Signal Transduction PathwayFinder
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Sample & Assay Technologies Gene expression analysis: JAK/STAT signaling
Background: Human parvovirus B19 can cause inflammatory cardiomyopathy� The viral protein NS1 may play a role in this pathophysiological process
Hypothesis: JAK/STAT signaling is important for immune and inflammatory processes, and may be involved in B19 infection.
Methods: Infect endothelial cells (HMEC-1) with NS1. Examine STAT activation and target gene expression
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Sample & Assay Technologies Gene expression analysis: JAK/STAT signaling
Conclusions: Upregulation of STAT3 signaling by NS1 may play a role in the mechanism for viral evasion of the immune response.
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Sample & Assay Technologies Signal transduction experimental design
Gene expression� RT-PCR
Epigenetics� miRNA� DNA methylation� Histone modifications
Functional studies� Transcription factor reporter assays� siRNA/shRNA
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QIAGEN offers a variety of products for signal transduction
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Sample & Assay Technologies Epigenetics overview
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Structural gene NFκB BS
Activated transcription factors
p53 BS
p53
NFκB
Protein “A”
mRNA ”A”
MeMeMe
Histones
Histone-DNA interactions
DNA methylation
Me AcMe Me MeMe
DNA methylation
miRNAshRNAsiRNA
Transcription initiation complex
+
–
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Sample & Assay Technologies miRNA products from QIAGEN
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3’ UTR reportersmiRNA Targets Arrays
Target identification Expression
Isolation
Function
miRNA mimics & inhibitors
miScript miRNA PCR Array & Assays
miRNeasy
miRNAstudies
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Sample & Assay Technologies miRNA: WNT signaling
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Background: What is the molecular mechanism for TIMP-1 regulation in mesenchymal stem cells?
Methods: Knockdown TIMP-1 and identify differentially-expressed miRNAs via miFindermiScript PCR Array. Confirm WNT signaling genes are targets.
Results: WNT signaling is identified as a part of TIMP-1 regulation. Hsa-let-7f is shown as a link between TIMP-1 and WNT signaling.
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Sample & Assay Technologies miRNA: WNT signaling
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miFinder
3’-UTR reporter
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Sample & Assay Technologies Epigenetics overview
Signal transduction targets, Frederick, MD, 3/15/13 29
Structural gene NFκB BS
Activated transcription factors
p53 BS
p53
NFκB
Protein “A”
mRNA ”A”
MeMeMe
Histones
Histone-DNA interactions
DNA methylation
Me AcMe Me MeMe
DNA methylation
miRNAshRNAsiRNA
Transcription initiation complex
+
–
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Sample & Assay Technologies Pyrosequencing
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Sample & Assay Technologies DNA methylation: WNT signaling
Hypothesis: SFRP1 expression is commonly lost during renal cancer due to hypermethylation. Can SFRP1 methylation correlate with renal cancer risk?
Methods: SFRP1 methylation analysis of age-matched renal cancer samples via pyrosequencing.
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Sample & Assay Technologies DNA methylation: WNT signaling
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Sample & Assay Technologies DNA methylation: WNT signaling
Hypothesis: SFRP1 expression is commonly lost during renal cancer due to hypermethylation. Can SFRP1 methylation correlate with renal cancer risk?
Methods: SFRP1 methylation analysis of age-matched renal cancer samples via pyrosequencing.
Conclusion: SFRP1 hypermethylation increases with age and correlates with cancer risk.
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Sample & Assay Technologies Epigenetics overview
Signal transduction targets, Frederick, MD, 3/15/13 34
Structural gene NFκB BS
Activated transcription factors
p53 BS
p53
NFκB
Protein “A”
mRNA ”A”
MeMeMe
Histones
Histone-DNA interactions
DNA methylation
Me AcMe Me MeMe
DNA methylation
miRNAshRNAsiRNA
Transcription initiation complex
+
–
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Sample & Assay Technologies Histone ChIP: TGFβ signaling
Background: What are the epigenetic alterations during Ras-induced cellular senescence?
Methods: Genome-wide gene expression by microarray. Histone modifications analyzed by genome-wide ChIP-seq and correlated with gene expression.� Active: H3K4me3� Repressed: H3K27me3
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Sample & Assay Technologies Histone ChIP: TGFβ signaling
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Active
Repressed
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Sample & Assay Technologies Histone ChIP: TGFβ signaling
Background: What are the epigenetic alterations during Ras-induced cellular senescence?
Methods: Genome-wide gene expression by microarray. Histone modifications analyzed by genome-wide ChIP-seq.
Conclusions: TGFβ signaling is involved in Ras-induced senescence
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Sample & Assay Technologies Signal transduction experimental design
Gene expression� RT-PCR
Epigenetics� miRNA� DNA methylation� Histone modifications
Functional studies� Transcription factor reporter assays� siRNA/shRNA
Signal transduction targets, Frederick, MD, 3/15/13 38
QIAGEN offers a variety of products for signal transduction
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Sample & Assay Technologies Reporter assay overview
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Sample & Assay Technologies Epigenetics overview
Signal transduction targets, Frederick, MD, 3/15/13 40
Structural gene NFκB BS
Activated transcription factors
p53 BS
p53
NFκB
Protein “A”
mRNA ”A”
MeMeMe
Histones
Histone-DNA interactions
DNA methylation
Me AcMe Me MeMe
DNA methylation
miRNAshRNAsiRNA
Transcription initiation complex
+
–
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Sample & Assay Technologies Reporter assay & shRNA: Notch signaling
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Background: Luminal breast cancer (ER+, PR+) is common and has a favorable prognosis� Antiestrogen therapy is a common treatment� Relapses often include “basal” breast cancer (ER-, PR-), which has a poor prognosis
Hypothesis: Luminal breast cancers often include a subset of basal breast cancer cells. � This subset grows rapidly during antiestrogen treatment
Methods: Develop breast cancer cell lines with progressive luminal to basal characteristics� Microarray gene expression analysis
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Sample & Assay Technologies Reporter assay & shRNA: Notch signaling
Results: Notch signaling is commonly dysregulated in basal breast cancer cells� Activated during anti-estrogen therapy� Inhibited by γ-secretase inhibitors
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Sample & Assay Technologies Reporter assay & shRNA: Notch signaling
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Reporter assay
Luminal
Luminobasal
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Sample & Assay Technologies Reporter assay & shRNA: Notch signaling
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Hypothesis: Luminal breast cancers often include a subset of basal breast cancer cells. � This subset grows rapidly during antiestrogen treatment
Methods: Develop breast cancer cell lines with progressive luminal to basal characteristics� Microarray gene expression analysis
Conclusions: Luminal breast cancer should be treated with anti-estrogens plus γ-secretaseinhibitors
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Sample & Assay Technologies Conclusions
Many signal transduction pathways� Typical receptor to target gene
� TGFβ� WNT
� Transcription factor-focused � JAK/STAT� p53
QIAGEN offers many methods to study these cellular processes� Gene expression
� RT2 Profiler PCR Arrays & Assays� Epigenetics
� miScript miRNA PCR System� EpiTect Methyl II PCR Arrays & Assays� EpiTect ChIP qPCR Arrays & Assays
� Functional studies� Cignal Reporter Assays� SureSilencing shRNA Plasmid� Flexitube/Flexiplate siRNA, GeneSolution, Premix, AllStar
Control
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Sample & Assay Technologies Thank you for attending!
Signal transduction targets, Frederick, MD, 3/15/13 46
(US and Canada only)
PCR Array Starter Pack- Promotion Code FDK-PAFAS22
PCR Arrays of any Pathway (FREE)� 2 96-well/100-well (2 samples) OR 1 384-well (4 samples) Required Reagents (w/ Purchase)� RT2 First-Strand cDNA Synthesis Kit� RT2 SYBR Green Mastermix (2-Pack)
Email: [email protected] OR [email protected]
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