This document provides an overview of Wnt signaling and research tools for studying the pathway. It begins with an introduction to Wnt proteins and receptors, and describes the canonical and noncanonical Wnt signaling pathways. The document then discusses the function of Wnt signaling in development, tissue homeostasis, and various pathological conditions. Various research tools for investigating Wnt signaling are presented, including whole genome expression profiling, siRNA inhibition of β-catenin, Wnt target gene PCR arrays, and EpiTech methylation arrays. The document proposes a workflow for developing a Wnt gene expression signature and demonstrates using custom PCR arrays and methylation analysis to understand differential Wnt responses between cell lines. It concludes by describing a combined Wnt signaling PCR
Clinical molecular diagnostics for drug guidanceNikesh Shah
1. Be familiar with next generation molecular diagnostic techniques that can provide guidance in clinical decision making
2. Identify the utility of these diagnostic approaches with some examples
3. Be aware of the challenges that exist in implementing these tools as part of the routine clinical decision making process, especially in resource limited settings
Clinical molecular diagnostics for drug guidanceNikesh Shah
1. Be familiar with next generation molecular diagnostic techniques that can provide guidance in clinical decision making
2. Identify the utility of these diagnostic approaches with some examples
3. Be aware of the challenges that exist in implementing these tools as part of the routine clinical decision making process, especially in resource limited settings
Postdoctoral work on Vangl2, a protein involved in cell polarity. Cell polarity is the capability of a cell to be able to orient itself in a particular direction.
Developing a Rapid Clinical Sequencing System to Classify Meningioma: Meet th...QIAGEN
Meningioma’s display a broad spectrum of clinical, histological and cytogenetic features even within the same WHO grade often posing a challenge for classification and prognostic stratification. In this webinar, we will describe our experience of using targeted amplicon sequencing to develop rapid clinical sequencing system to identify and confirm the meningioma genotype in just two weeks. In addition the details of the three meningioma categories and the genes involved will be discussed.
New technology and workflow for integrated collection, stabilization and puri...QIAGEN
Research into non-invasive prenatal testing (NIPT) and circulating tumor DNA (ctDNA) testing based on circulating cell-free DNA (ccfDNA) is rapidly expanding. However, detection and quantification of ccfDNA is compromised by the release of genomic DNA (gDNA) from lymphocytes due to mechanical lysis or apoptosis during blood collection, storage and transport. PreAnalytiX has developed the PAXgene Blood ccfDNA System, consisting of the PAXgene Blood ccfDNA Tube, a plastic blood collection tube with a unique, non-crosslinking chemistry that preserves extracellular levels of ccfDNA and prevents the release of intracellular DNA from cells into the plasma, and the QIAsymphonyPAXgene Blood ccfDNA Kit for automated ccfDNA extraction from up to 5 ml of plasma. In this slidedeck, this new technology development is presented in comparison to other existing technologies.
Dr. Ben Hause - Metagenomic Sequencing for Virus Discovery and CharacterizationJohn Blue
Metagenomic Sequencing for Virus Discovery and Characterization - Dr. Ben Hause, Kansas State University, from the 2015 North American PRRS Symposium, December 4 - 5, 2015, Chicago, IL, USA.
More presentations at http://www.swinecast.com/2015-north-american-prrs-symposium
This is the Powerpoint presentation from my recent presentation at the TTP LabTech US Acumen Users Group Meeting (UGM) held at the British Consulate-General in Cambridge, MA on May 18, 2010
Presentation from the ECDC expert consultation on Whole Genome Sequencing organised by the European Centre of Disease Prevention and Control - Stockholm, 19 November 2015
Kinases belong to the family of phosphotransferases and play critical roles in the process known as phosphorylation in which a phosphorylated substrate and ADP are produced.
https://www.creative-biogene.com/products/kinase-stable-cell-lines.html
Moving Towards a Validated High Throughput Sequencing Solution for Human Iden...Thermo Fisher Scientific
Presented by Jennifer D. Churchill, PhD during a special Lunch and Learn session during the American Academy of Forensic Sciences (AAFS) 67th annual conference, February 2015. / Conclusions
• Robust panels of identity and ancestry SNPs
• Robust STR panel
• Whole genome mtDNA sequencing
• Highly informative
• Sensitive
• Quantitative – scaling comparison
• Low density chip is not necessarily a bad chip
• Wide range of density can still yield high quality data
• Based on results continue development and validation
Cell-based Reporter Assays: Measure 45 Signaling Pathway Activity in Any Cel...Qiagen - Egypt
Would you like to measure signaling pathway activity in your favorite cell? Learn how to successfully apply convenient and robust reporter assays to your RNA interference, gene over-expression, protein, or small molecule studies. The Cignal Reporter Assays are an excellent tool for studying pathway signaling activity in cells that are amenable to transfection, available for studying numerous pathways including (ROS, Wnt, NF-kB, Notch, cAMP/PKA, TGFbeta, and the Cignal Lenti Reporter Assays combines the power of a lentiviral delivery system with our robust transcription factor reporter technology, enabling you to study signal pathways in virtually any cell type. You can find a technology overview, protocol tutorial, and application examples in the following presentation.
Cell-based Reporter Assays: Measure 45 Signaling Pathway Activity in Any Cell...QIAGEN
Would you like to measure signaling pathway activity in your favorite cell? Learn how to successfully apply convenient and robust reporter assays to your RNA interference, gene over-expression, protein, or small molecule studies. The Cignal Reporter Assays are an excellent tool for studying pathway signaling activity in cells that are amenable to transfection, available for studying numerous pathways including (ROS, Wnt, NF-kB, Notch, cAMP/PKA, TGFbeta, and the Cignal Lenti Reporter Assays combines the power of a lentiviral delivery system with our robust transcription factor reporter technology, enabling you to study signal pathways in virtually any cell type. You can find a technology overview, protocol tutorial, and application examples in the following presentation.
Postdoctoral work on Vangl2, a protein involved in cell polarity. Cell polarity is the capability of a cell to be able to orient itself in a particular direction.
Developing a Rapid Clinical Sequencing System to Classify Meningioma: Meet th...QIAGEN
Meningioma’s display a broad spectrum of clinical, histological and cytogenetic features even within the same WHO grade often posing a challenge for classification and prognostic stratification. In this webinar, we will describe our experience of using targeted amplicon sequencing to develop rapid clinical sequencing system to identify and confirm the meningioma genotype in just two weeks. In addition the details of the three meningioma categories and the genes involved will be discussed.
New technology and workflow for integrated collection, stabilization and puri...QIAGEN
Research into non-invasive prenatal testing (NIPT) and circulating tumor DNA (ctDNA) testing based on circulating cell-free DNA (ccfDNA) is rapidly expanding. However, detection and quantification of ccfDNA is compromised by the release of genomic DNA (gDNA) from lymphocytes due to mechanical lysis or apoptosis during blood collection, storage and transport. PreAnalytiX has developed the PAXgene Blood ccfDNA System, consisting of the PAXgene Blood ccfDNA Tube, a plastic blood collection tube with a unique, non-crosslinking chemistry that preserves extracellular levels of ccfDNA and prevents the release of intracellular DNA from cells into the plasma, and the QIAsymphonyPAXgene Blood ccfDNA Kit for automated ccfDNA extraction from up to 5 ml of plasma. In this slidedeck, this new technology development is presented in comparison to other existing technologies.
Dr. Ben Hause - Metagenomic Sequencing for Virus Discovery and CharacterizationJohn Blue
Metagenomic Sequencing for Virus Discovery and Characterization - Dr. Ben Hause, Kansas State University, from the 2015 North American PRRS Symposium, December 4 - 5, 2015, Chicago, IL, USA.
More presentations at http://www.swinecast.com/2015-north-american-prrs-symposium
This is the Powerpoint presentation from my recent presentation at the TTP LabTech US Acumen Users Group Meeting (UGM) held at the British Consulate-General in Cambridge, MA on May 18, 2010
Presentation from the ECDC expert consultation on Whole Genome Sequencing organised by the European Centre of Disease Prevention and Control - Stockholm, 19 November 2015
Kinases belong to the family of phosphotransferases and play critical roles in the process known as phosphorylation in which a phosphorylated substrate and ADP are produced.
https://www.creative-biogene.com/products/kinase-stable-cell-lines.html
Moving Towards a Validated High Throughput Sequencing Solution for Human Iden...Thermo Fisher Scientific
Presented by Jennifer D. Churchill, PhD during a special Lunch and Learn session during the American Academy of Forensic Sciences (AAFS) 67th annual conference, February 2015. / Conclusions
• Robust panels of identity and ancestry SNPs
• Robust STR panel
• Whole genome mtDNA sequencing
• Highly informative
• Sensitive
• Quantitative – scaling comparison
• Low density chip is not necessarily a bad chip
• Wide range of density can still yield high quality data
• Based on results continue development and validation
Cell-based Reporter Assays: Measure 45 Signaling Pathway Activity in Any Cel...Qiagen - Egypt
Would you like to measure signaling pathway activity in your favorite cell? Learn how to successfully apply convenient and robust reporter assays to your RNA interference, gene over-expression, protein, or small molecule studies. The Cignal Reporter Assays are an excellent tool for studying pathway signaling activity in cells that are amenable to transfection, available for studying numerous pathways including (ROS, Wnt, NF-kB, Notch, cAMP/PKA, TGFbeta, and the Cignal Lenti Reporter Assays combines the power of a lentiviral delivery system with our robust transcription factor reporter technology, enabling you to study signal pathways in virtually any cell type. You can find a technology overview, protocol tutorial, and application examples in the following presentation.
Cell-based Reporter Assays: Measure 45 Signaling Pathway Activity in Any Cell...QIAGEN
Would you like to measure signaling pathway activity in your favorite cell? Learn how to successfully apply convenient and robust reporter assays to your RNA interference, gene over-expression, protein, or small molecule studies. The Cignal Reporter Assays are an excellent tool for studying pathway signaling activity in cells that are amenable to transfection, available for studying numerous pathways including (ROS, Wnt, NF-kB, Notch, cAMP/PKA, TGFbeta, and the Cignal Lenti Reporter Assays combines the power of a lentiviral delivery system with our robust transcription factor reporter technology, enabling you to study signal pathways in virtually any cell type. You can find a technology overview, protocol tutorial, and application examples in the following presentation.
Total RNA Discovery for RNA Biomarker Development WebinarQIAGEN
Precision medicine offers to transform patient care by targeting treatment to those with most to gain. To date the most significant advances have been at the level of DNA, for example, the use of somatic DNA alterations as diagnostic indicators of disease and for prediction of pharmacodynamic response. Development of RNA expression signatures as biomarkers has been more problematic. While RNA expression analysis has yielded valuable insights into the biological mechanisms of disease, RNA is a more unstable molecule than DNA, and more easily damaged or degraded during sample collection and isolation. In addition, RNA levels are inherently dynamic and gene expression signatures are extraordinarily complex. Recently, much progress has been made in identifying key changes in gene expression in cancer and other diseases, as well as identifying expression signatures in circulating nucleic acid that have the potential to be developed into diagnostic and prognostic indicators.
Please note: This presentation accompanies a recorded webinar at:
https://www1.gotomeeting.com/register/347794241
Biomarkers for studying gene regulation and cell function can be efficiently analyzed by multiplexed methods. Dr. Jim Lazar from OriGene Technologies will provide an overview of four different but related detection technologies that can be used to analyze genetic variants, microRNA expression, transcription factor binding, and protein expression on the Luminex xMAP platform. OriGene’s broad panel of assays and tools for discovery, analysis and validation of multiple classes of important biomarkers will allow researcher to develop more accurate descriptions of biologically complex systems.
I received a PhD in April of 2007 from the Schultz Lab at the Scripps Research Institute in La Jolla, CA. Here is a PowerPoint presentation of my primary work - a use of functional genomics tools to probe cellular disease problems, notably in cancer models.
Spinach ™ splice sensor a fluorescent drug screening platformBetty Cosgrove
Lucerna, Inc:
Lucerna develops unique RNA-based research tools to detect intractable biomolecules for basic research, industrial, and drug discovery applications. We are a team of experts in the field of RNA biology and the founders who invented a proprietary fluorescent aptamer Spinach™ technology.
1. Sample & Assay Technologies
Wnt Signaling: New research solutions
Zhong Wu, Ph.D.
R&D Scientist
2. Sample & Assay Technologies
Outline
Overview of Wnt signaling
Function of Wnt signaling
Research tools to study Wnt signaling
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3. Sample & Assay Technologies
Outline
Overview of Wnt signaling
Wnt discovery and family members
Canonical
Noncanonical
Function of Wnt signaling
Research tools to study Wnt signaling
3
4. Sample & Assay Technologies
Wnts and receptors
Discovery of Wnt
Murine oncogene Int-1
Homolog to Drosophila segment polarity gene
Wingless
Wnt proteins
19 members
Highly conserved
Secreted glycoproteins
Activate cell surface receptors
Wnt receptors
FZD/LRP receptor complex
10 members of FZD
LRP5 and LRP6 coreceptor
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5. Sample & Assay Technologies
Different Wnt signaling pathways
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9. Sample & Assay Technologies
Topics to cover
Overview of Wnt signaling
Wnt discovery and family members
Canonical
Noncanonical
Function of Wnt signaling
Development
Tissue homeostasis
Pathological condition
Research tools to study Wnt signaling
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10. Sample & Assay Technologies
Wnt signaling in embryonic development
Function during development
Cell fate determination
Proliferation
Migration, motility and polarity specification
Tissue patterning
Organogenesis and organization of the body plan
Almost every development process
Axial formation
Mesoderm specification
Production of presomitic mesoderm
Direct patterning of the nervous system
Suppress chondrogenic potential of joint interzone
cells
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11. Sample & Assay Technologies
Wnt signaling in tissue renewal
Zebrafish tail fin and heart regeneration
Maintain stem-cell like fate
Intestinal epithelium
Skin
Hematopoietic cells
Neural stem cells
Nature 434, 843-850 (2005) & Development 134, 479-489 (2007)
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12. Sample & Assay Technologies
Wnt signaling in developmental malformation
Development cell 17,9-26 (2009)
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13. Sample & Assay Technologies
Wnt signaling in cancer
Eur J Cancer 45,2759-2767 (2009), Nature 434, 843-850 (2005) & Wnt homepage
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14. Sample & Assay Technologies
Wnt signaling in CNS-related diseases
Alzheimer’s disease
Reduced β-catenin signaling
Tau hyperphosphorylation by GSK3β
LRP6 association
Parkinson’s disease
Wnt-β-catenin and Wnt-PCP
Dopaminergic development and
neurogenesis
Schizophrenia
Wnt1 overexpression
SNPs in FZ3
DISC1 interacts with GSK3β
Nat Rev Neurosci. 2010;11(2):77-86
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15. Sample & Assay Technologies
Wnt signaling in other pathological condition
High bone mass and Osteoporosis
LRP5 activating (LRP5G171V)
LRP5 lose of function mutation
Type II diabetes
Wnt5B
TCF7L2 (TCF4)
Pulmonary fibrosis
Activated β-catenin signaling
N Engl J Med 2002;346:1513-1521, Am J Hum Genet, 2004,75:832-43
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16. Sample & Assay Technologies
Topics to cover
Overview of Wnt signaling
Wnt discovery and family members
Canonical
Noncanonical
Function of Wnt signaling
Development
Tissue homeostasis
Pathological condition
Research tools to study Wnt signaling
A real case study about Wnt pathway activity
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17. Sample & Assay Technologies
Where to measure pathway activity?
physiological
pathological
WNT
WNT
Fzd
Fzd
L
R
P
Dsh
Pathway
Activity?
APC Axin
GSK3β
β
P
TCF/
LEF1
Dsh
APC
Axin
GSK3β
β
P
β -catenin
β -catenin
L
R
P
β -catenin
β -catenin
Myc
TCF/
LEF1
CCND1
Myc
CCND1
Downstream effect!
Traditional indicators provide insufficient information of the regulatory status of the whole
pathway signaling
Multi-gene expression based methods measure pathway alteration as a function of the
downstream effect of pathway regulation on multiple gene expression changes
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18. Sample & Assay Technologies
Wnt
A simple way to measure downstream effects
Notch
NFκB
κ
TGFβ
β
IL6/STAT3
Signature gene
gene expression change
measure by real-time PCR
Specific gene panel with companion score to monitor individual pathway activity
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19. Sample & Assay Technologies
Proposed work flow for Wnt gene expression signature
development
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20. Sample & Assay Technologies
Experiment setup for whole genome gene expression profiling
Whole genome gene expression profiling after altering Wnt/β-catenin pathway
activity
Illumina bead chip analysis by SABio Service Core
Treatment
Stimulation – Wnt3a
Inhibition – siRNA for β-catenin (Qiagen FlexiTube siRNA)
Wnt Pathway activity readout
Wnt target genes (Wnt signaling target PCR array)
Active β-catenin protein levels
Cell lines
293H
FHC
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21. Sample & Assay Technologies
QIAGEN-SABiosciences service core
Complete service for genomic and expression analysis that
delivers robust and reproducible results
Whole genome
Illumina expression profiling
Illumina genotyping
Pathway/focused panel
Mutation profiling
Methylation
PCR array
miRNA PCR array
Individual gene/Locus
Mutation
Methylation
qRT-PCR SYBR Green
Sample prep
Cell, tissue or biofluids
Fixed tissue
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24. Sample & Assay Technologies
Experiment setup for whole genome gene expression profiling
Whole genome gene expression profiling after altering Wnt/ β-catenin pathway
activity
Illumina bead chip analysis by SABio Service Core
Treatment
Stimulation – Wnt3a
Inhibition – siRNA for β-catenin (Qiagen FlexiTube siRNA)
Wnt Pathway activity readout
Wnt target genes (Wnt signaling target PCR array)
Active β-catenin protein levels
Cell lines
293H
FHC
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25. Sample & Assay Technologies
What is Wnt signaling target PCR Array?
• 84 genes responsive to
Wnt signaling pathway
• 5 Housekeeping Genes
• Genomic DNA
Contamination Control
• Reverse Transcription
Controls (RTC) n=3
• Positive PCR Controls
(PPC) n=3
Supported PCR Array Formats
96-Well Plate
384-Well Plate
100-Well Disc
96x96 Chip
Over 100 different pathways available!
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26. Sample & Assay Technologies
control
RT2 PCR Arrays Work Flow
experimental
cDNA Synthesis (C-03 kit)
Genomic DNA Removal Step (5 min.)
Reverse Transcription Step (20 min.)
Load Plates
1 Sample per PCR Array
2 minutes with multi-channel pipet
Run 40 cycle qPCR Program
Standard cycling conditions for all Real
Time PCR Instruments
2 hours
Upload and Analyze Data (FREE)
15 minutes from Raw Ct to
Fold Change Data
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27. Sample & Assay Technologies
Experiment setup for whole genome gene expression profiling
Whole genome gene expression profiling after altering Wnt/ β-catenin pathway
activity
Illumina bead chip analysis by SABio Service Core
Treatment
Stimulation – Wnt3a
Inhibition – siRNA for β-catenin (Qiagen FlexiTube siRNA)
Wnt Pathway activity readout
Wnt target genes (Wnt signaling target PCR array)
Active β-catenin protein levels
Cell lines
293H
FHC
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29. Sample & Assay Technologies
Experiment problem and approach
Q: Why the effect of Wnt stimulation on FHC cells was
weaker than that on 293H cells?
Hypothesis: Defect in Wnt signaling pathway transduction in
FHC cell?
Approach: Expression profiling of 293H and FHC cells on
Wnt pathway genes with Wnt Signaling Pathway PCR
array.
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35. Sample & Assay Technologies
Interesting question during experiment
wnt3a
X
Y
LEF1
Q: What’s the mechanism involved in differential Wnt
target gene expression between HepG2 and IMR90 cells?
Hypothesis: Epigenetic silencing of Wnt pathway
transducers in HepG2 cell?
Approach: Examine methylation status of Wnt pathway
transduction genes with Wnt signaling EpiTech Methyl II
qPCR array.
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36. Sample & Assay Technologies
EpiTech Methyl II qPCR Array Protocol Overview
Innovative technology enabling fast and accurate detection of
CpG island DNA methylation of individual genes, and disease or
pathway focused gene panels
(0.5 – 4 ug of Genomic DNA)
Raw Ct
Values
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43. Sample & Assay Technologies
Pathway activity score
Summary of Pathway Activity Scores
Sample
Pathway activity score
P value
value*
A
-0.925142
0.01
B
0.904624
0.01
C
0.146092
0.82
Significant call of pathway activity change
P < 0.05
Score >0.3 or <-0.3
Available Plus and Predictor arrays
Wnt
NFκB
IL6/STAT3
Notch
TGFβ
Oxidative stress
ER stress
Mitochondrial
Hypoxia
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44. Sample & Assay Technologies
Application example: cancer stem cell analysis with Wnt
Pathway Plus Array
Wnt pathway activity confers chemoresistance to cancer
stem-like cells in a neuroblastoma cell line
CD133-
Tumour Biol. 2012 Aug 11. [Epub ahead of print]
Wnt Signaling RT2 PCR Array Plus uncovered:
Differential Wnt pathway gene expression profiles
associated with CD133+ (stem like) and CD133neuroblastoma cells
CD133+
Activation of Wnt pathway signaling in CD133+ cells
(sample 1) while repression of Wnt signaling in
CD133- cells (sample 3) upon Dox treatment.
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45. Sample & Assay Technologies
Application example: neural stem cell analysis with RT2
Predictor PCR Arrays
NSC (undifferentiated)
NSC (differentiated 10d)
Notch
Wnt
IL6/STAT3
NFkB
TGFβ
RT2 Predictor array indicted activation of Notch and TGFβ pathway activity while
repression of Wnt signaling during neural stem cell differentiation process
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46. Sample & Assay Technologies
Available Plus and Predictor arrays
RT2 Profiler Plus PCR Array
Human WNT Signaling Pathway Plus
Human IL6/STAT3 Signaling Pathway Plus
Human TGFb/BMP Signaling Pathway Plus
Human Notch Signaling Pathway Plus
Human NFkB Signaling Pathway Plus
Human Oxidative Stress Plus
Human Hypoxia Signaling Pathway Plus
Human Unfolded Protein Response Plus
Human Mitochondrial Energy Metabolism Plus
RT2 Predictor PCR Array
Human WNT Signaling Pathway Activity
Human IL6/STAT3 Signaling Pathway Activity
Human TGFb Signaling Pathway Activity
Human Notch Signaling Pathway Activity
Human NFkB Signaling Pathway Activity
Human Oxidative Stress Toxicity
Human Hypoxia Signaling Pathway Activity
Human Unfolded Protein Response Toxicity
Human Mitochondrial Energy Metabolism Toxicity
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47. Sample & Assay Technologies
Summary
Overview of Wnt signaling
Wnt discovery and family members
Canonical - β-catenin
Nonncanonical – Wnt/JNK & Wnt/Ca2+
Function of Wnt signaling
Development – many aspects
Tissue homeostasis – maintain stem cell like feature
Pathological condition – cancer, bone, nervous
Wnt gene signature development with various tools
– A gene expression signature to measure Wnt pathway activity
RT2 Profile PCR array – study gene expression changes related to Wnt
signaling
siRNA – gene silencing for functional Wnt pathway study
EpiTech Methyl array – explore mechanism of gene expression changes
RT2 Predictor PCR array – assess pathway activity with real-time PCR
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48. Sample & Assay Technologies
Additional Resources
Questions?
Contact Technical Support
9 AM – 6 PM Eastern M – F
Contact: 1-888-503-3187 OR
support@SABiosciences.com
QIAWEBINARS@QIAGEN.COM
E-Learning Center:
Recorded Seminars
48