This document discusses signal transduction pathways and provides an overview of products from QIAGEN for their analysis. It describes several key signaling pathways including TGFβ, WNT, Notch, p53, and JAK/STAT. It outlines methods for studying these pathways including gene expression analysis using RT-PCR and epigenetic techniques like miRNA, DNA methylation, and histone ChIP analysis. Reporter assays and RNAi tools for functional studies are also discussed. Examples are provided showing how these techniques have been applied to study topics like prostate cancer, viral infection, and breast cancer.

1. Sample & Assay Technologies
Signal transduction targets:
Research solutions for pathway analysis
Jennifer Gibbons, Ph.D.
R&D Scientist

2. Sample & Assay Technologies
Biology-focused solutions for pathway analysis
• SABiosciences is now a
Company
Signal transduction targets, Frederick, MD, 3/15/13
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Background: Signal transduction
The process of disseminating an extracellular signal, resulting in a
functional response
Ligand
Receptor
Downstream
Signaling
Transcription
Factor
Target Gene
Expression
Biological
Function
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4. 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
Signal transduction targets, Frederick, MD, 3/15/13
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5. 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
Signal transduction targets, Frederick, MD, 3/15/13
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6. Sample & Assay Technologies
TGFβ superfamily signaling pathways
BMP
TGFβ
Activin
Nodal
http://www.genome.jp/kegg/pathway/hsa/hsa04350.html
Signal transduction targets, Frederick, MD, 3/15/13
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7. Sample & Assay Technologies
Ligands
TGFβ superfamily signaling pathways
Receptors
Signaling/TFs
Target Genes
http://www.genome.jp/kegg/pathway/hsa/hsa04350.html
Signal transduction targets, Frederick, MD, 3/15/13
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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
Signal transduction targets, Frederick, MD, 3/15/13
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9. Sample & Assay Technologies
WNT signaling pathways
Canonical
PCP
WNT/Ca2+
http://www.genome.jp/kegg/pathway/hsa/hsa04310.html
Signal transduction targets, Frederick, MD, 3/15/13
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10. Sample & Assay Technologies
WNT signaling pathways
Ligands Receptors
Downstream
Signaling
TFs
Target
Genes
http://www.genome.jp/kegg/pathway/hsa/hsa04310.html
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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
Signal transduction targets, Frederick, MD, 3/15/13
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12. Sample & Assay Technologies
Notch signaling pathway
Ligands/receptors/cofactors
TFs
Target
genes
http://www.genome.jp/kegg/pathway/hsa/hsa04330.html
Signal transduction targets, Frederick, MD, 3/15/13
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p53 signaling pathway
http://www.genome.jp/kegg/pathway/hsa/hsa04115.html
Signal transduction targets, Frederick, MD, 3/15/13
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14. Sample & Assay Technologies
JAK/STAT signaling pathway
Signal transduction targets, Frederick, MD, 3/15/13
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15. Sample & Assay Technologies
Signal transduction experimental design
QIAGEN offers a variety of products for signal transduction
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
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16. Sample & Assay Technologies
Principles of qRT-PCR: Overview
Real-time PCR
Amplify and simultaneously quantify target DNA
Reverse transcription real-time PCR
Amplify and simultaneously quantify mRNA
Ct values: Threshold cycle
Signal transduction targets, Frederick, MD, 3/15/13
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17. Sample & Assay Technologies
Gene expression: Which pathway to choose?
Focus your research with the RT2 Profiler Signal Transduction
PathwayFinder
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)
Signal transduction targets, Frederick, MD, 3/15/13
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18. Sample & Assay Technologies
Mouse Signal Transduction PathwayFinder
Functional gene groupings identify what pathways may be affected
in your experimental system
Signal transduction targets, Frederick, MD, 3/15/13
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19. Sample & Assay Technologies
Western blots confirm PCR array results
Signal transduction targets, Frederick, MD, 3/15/13
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Maybe WNT signaling involved?
Signal transduction targets, Frederick, MD, 3/15/13
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21. Sample & Assay Technologies
Gene expression: Which pathway to choose?
Focus your research with the RT2 Profiler Signal Transduction
PathwayFinder
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.
Signal transduction targets, Frederick, MD, 3/15/13
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22. 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
Signal transduction targets, Frederick, MD, 3/15/13
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23. 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.
Signal transduction targets, Frederick, MD, 3/15/13
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24. Sample & Assay Technologies
Signal transduction experimental design
QIAGEN offers a variety of products for signal transduction
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
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25. Epigenetics overview
Sample & Assay Technologies
Activated
transcription factors
miRNA
shRNA
siRNA
Protein “A”
NFκB
+
p53
Transcription
initiation complex
mRNA ”A”
–
Histones
p53 BS Me
Me
Me
Me Me
NFκB BS
DNA methylation
Histone-DNA
interactions
Ac
Structural gene
Me
Me Me
DNA methylation
Signal transduction targets, Frederick, MD, 3/15/13
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26. Sample & Assay Technologies
miRNA products from QIAGEN
miRNeasy
Isolation
Target identification
miRNA
studies
Expression
miScript miRNA PCR
Array & Assays
3’ UTR reporters
miRNA Targets Arrays
Function
miRNA mimics & inhibitors
Signal transduction targets, Frederick, MD, 3/15/13
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27. Sample & Assay Technologies
miRNA: WNT signaling
Background: What is the molecular mechanism for TIMP-1 regulation in mesenchymal stem
cells?
Methods: Knockdown TIMP-1 and identify differentially-expressed miRNAs via miFinder
miScript 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.
Signal transduction targets, Frederick, MD, 3/15/13
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28. Sample & Assay Technologies
miRNA: WNT signaling
miFinder
3’-UTR reporter
Signal transduction targets, Frederick, MD, 3/15/13
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29. Epigenetics overview
Sample & Assay Technologies
Activated
transcription factors
miRNA
shRNA
siRNA
Protein “A”
NFκB
+
p53
Transcription
initiation complex
mRNA ”A”
–
Histones
p53 BS Me
Me
Me
Me Me
NFκB BS
DNA methylation
Histone-DNA
interactions
Ac
Structural gene
Me
Me Me
DNA methylation
Signal transduction targets, Frederick, MD, 3/15/13
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30. Sample & Assay Technologies
Pyrosequencing
Signal transduction targets, Frederick, MD, 3/15/13
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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.
Signal transduction targets, Frederick, MD, 3/15/13
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32. Sample & Assay Technologies
DNA methylation: WNT signaling
Signal transduction targets, Frederick, MD, 3/15/13
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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.
Signal transduction targets, Frederick, MD, 3/15/13
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34. Epigenetics overview
Sample & Assay Technologies
Activated
transcription factors
miRNA
shRNA
siRNA
Protein “A”
NFκB
+
p53
Transcription
initiation complex
mRNA ”A”
–
Histones
p53 BS Me
Me
Me
Me Me
NFκB BS
DNA methylation
Histone-DNA
interactions
Ac
Structural gene
Me
Me Me
DNA methylation
Signal transduction targets, Frederick, MD, 3/15/13
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35. 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
Signal transduction targets, Frederick, MD, 3/15/13
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36. Sample & Assay Technologies
Histone ChIP: TGFβ signaling
Active
Repressed
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37. 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
Signal transduction targets, Frederick, MD, 3/15/13
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38. Sample & Assay Technologies
Signal transduction experimental design
QIAGEN offers a variety of products for signal transduction
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
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39. Sample & Assay Technologies
Reporter assay overview
Signal transduction targets, Frederick, MD, 3/15/13
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40. Epigenetics overview
Sample & Assay Technologies
Activated
transcription factors
miRNA
shRNA
siRNA
Protein “A”
NFκB
+
p53
Transcription
initiation complex
mRNA ”A”
–
Histones
p53 BS
Me
Me
Me
Me Me
NFκB BS
DNA methylation
Histone-DNA
interactions
Ac
Structural gene
Me
Me Me
DNA methylation
Signal transduction targets, Frederick, MD, 3/15/13
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41. Sample & Assay Technologies
Reporter assay & shRNA: Notch signaling
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
Signal transduction targets, Frederick, MD, 3/15/13
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42. 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
Signal transduction targets, Frederick, MD, 3/15/13
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43. Sample & Assay Technologies
Reporter assay & shRNA: Notch signaling
Reporter assay
Luminal
Luminobasal
Signal transduction targets, Frederick, MD, 3/15/13
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Reporter assay & shRNA: Notch signaling
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-secretase
inhibitors
Signal transduction targets, Frederick, MD, 3/15/13
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45. 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
Signal transduction targets, Frederick, MD, 3/15/13
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46. Sample & Assay Technologies
Thank you for attending!
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Required Reagents (w/ Purchase)
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RT2 SYBR Green Mastermix (2-Pack)
Email: Support@SABiosciences.com OR
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Call 1-888-503-3187 to order
(US and Canada only)
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