• Share
  • Email
  • Embed
  • Like
  • Save
  • Private Content
Signal transduction 2013
 

Signal transduction 2013

on

  • 324 views

 

Statistics

Views

Total Views
324
Views on SlideShare
324
Embed Views
0

Actions

Likes
0
Downloads
10
Comments
0

0 Embeds 0

No embeds

Accessibility

Categories

Upload Details

Uploaded via as Adobe PDF

Usage Rights

© All Rights Reserved

Report content

Flagged as inappropriate Flag as inappropriate
Flag as inappropriate

Select your reason for flagging this presentation as inappropriate.

Cancel
  • Full Name Full Name Comment goes here.
    Are you sure you want to
    Your message goes here
    Processing…
Post Comment
Edit your comment

    Signal transduction 2013 Signal transduction 2013 Presentation Transcript

    • Sample & Assay Technologies Signal transduction targets: Research solutions for pathway analysis Jennifer Gibbons, Ph.D. R&D Scientist
    • Sample & Assay Technologies Biology-focused solutions for pathway analysis • SABiosciences is now a Company Signal transduction targets, Frederick, MD, 3/15/13 2
    • Sample & Assay Technologies 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 Signal transduction targets, Frederick, MD, 3/15/13 3
    • 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 4
    • 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 5
    • 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 6
    • 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 7
    • 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 Signal transduction targets, Frederick, MD, 3/15/13 8
    • 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 9
    • Sample & Assay Technologies WNT signaling pathways Ligands Receptors Downstream Signaling TFs Target Genes http://www.genome.jp/kegg/pathway/hsa/hsa04310.html Signal transduction targets, Frederick, MD, 3/15/13 10
    • 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 Signal transduction targets, Frederick, MD, 3/15/13 11
    • 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 12
    • Sample & Assay Technologies p53 signaling pathway http://www.genome.jp/kegg/pathway/hsa/hsa04115.html Signal transduction targets, Frederick, MD, 3/15/13 13
    • Sample & Assay Technologies JAK/STAT signaling pathway Signal transduction targets, Frederick, MD, 3/15/13 14
    • 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 15
    • 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 16
    • 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 17
    • 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 18
    • Sample & Assay Technologies Western blots confirm PCR array results Signal transduction targets, Frederick, MD, 3/15/13 19
    • Sample & Assay Technologies Maybe WNT signaling involved? Signal transduction targets, Frederick, MD, 3/15/13 20
    • 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 21
    • 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 22
    • 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 23
    • 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 24
    • 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 25
    • 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 26
    • 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 27
    • Sample & Assay Technologies miRNA: WNT signaling miFinder 3’-UTR reporter Signal transduction targets, Frederick, MD, 3/15/13 28
    • 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 29
    • Sample & Assay Technologies Pyrosequencing Signal transduction targets, Frederick, MD, 3/15/13 30
    • 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. Signal transduction targets, Frederick, MD, 3/15/13 31
    • Sample & Assay Technologies DNA methylation: WNT signaling Signal transduction targets, Frederick, MD, 3/15/13 32
    • 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. Signal transduction targets, Frederick, MD, 3/15/13 33
    • 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 34
    • 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 35
    • Sample & Assay Technologies Histone ChIP: TGFβ signaling Active Repressed Signal transduction targets, Frederick, MD, 3/15/13 36
    • 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 37
    • 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 38
    • Sample & Assay Technologies Reporter assay overview Signal transduction targets, Frederick, MD, 3/15/13 39
    • 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 40
    • 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 41
    • 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 42
    • Sample & Assay Technologies Reporter assay & shRNA: Notch signaling Reporter assay Luminal Luminobasal Signal transduction targets, Frederick, MD, 3/15/13 43
    • Sample & Assay Technologies 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 44
    • 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 45
    • Sample & Assay Technologies Thank you for attending! 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: Support@SABiosciences.com OR QIAWEBINARS@QIAGEN.COM Call 1-888-503-3187 to order (US and Canada only) Signal transduction targets, Frederick, MD, 3/15/13 46