Toll-like receptors (TLRs) play an important role in the innate immune system and inflammation. TLRs recognize pathogen-associated molecular patterns and activate signaling pathways that induce inflammatory responses. This webinar discusses TLR signaling and the role of TLRs in inflammation, including their association with various diseases. It also explores how long non-coding RNAs and microRNAs help regulate TLR signaling pathways and the inflammatory response. The webinar promotes QIAGEN tools for studying TLRs, such as RT-PCR arrays, that can help profile gene and RNA expression changes related to TLR signaling.

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Toll-like Receptors in Inflammation
Samuel J. Rulli, Ph.D.
Samuel.Rulli@qiagen.com
Questions? Email us at:
BRCsupport@qiagen.com
QIAwebinars@qiagen.com
Part Two of a Four-Part Webinar Series on Microbiology

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A four-part webinar series on host responses
Toll-like receptorsin inflammation 2
Exploring the first line of defense: research tools for the innate
immune system
Toll-like receptors in inflammation
Studying the adaptive immune response: tools for T and B cell
research
The crosstalk between cancer inflammation and immunity:
exploring cancer immune responses
Explore host responses and defense mechanisms:
2
1
3
4

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Legal Disclaimer
Toll-like receptorsin inflammation 3
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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
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Agenda
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• Inflammation: the role of Toll-like receptors (TLRs)
• Toll-like receptors: an overview
• Toll-like receptors: signaling
• TLR8 in HIV expressionfrom latent reservoirs
◦ RT2 Profiler PCR Arrays
• lncRNAs mediate immune regulation
• TLR-mediated induction of microRNAs
◦ microRNAs associated with TLR signaling
◦ miScript miRNA PCR Arrays
• Summary

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Inflammation: an introduction
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̣Definition: a protective response to tissue damage or microbes that serves to
destroy, dilute or wall off the injuring agent and the injured tissues
• Cytokines & chemokines
• Signaling pathways
• Immune system composition
• Epigenetic changes
• mRNA changes
Microbes/
infection/
tissue damage
Acute
inflammation
Infection
clearance/
tissue
homeostasis
Chronic
inflammation
Pre-cancer
& cancer/
chronic
inflammatory
diseases
Toll-like
receptors
Toll-like
receptors

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Toll-like receptors: signaling
• Toll-like receptors (TLRs):
◦ A family of membrane-spanning innate immune
receptors
◦ Recognize ligands derived from bacteria, fungi,
viruses, parasites and host proteins/factors via
pathogen associated molecular patterns (PAMPs)
• All TLRs share a common modular structure:
◦ A leucine rich repeat (LRR)-containing ectodomain
responsible for ligand-binding
◦ A membrane-spanningregion
◦ A cytosolic signaling domain called the
Toll-interleukin-1 receptor homology domain (TIR)
• TLRs induce the expression of genes required for:
◦ The inflammatory response, including
▬ Inflammatory cytokines
▬ Chemokines
▬ Antimicrobial molecules
▬ Major histocompatibility (MHC)
▬ Co-stimulatory molecules important for adaptive
immune activation
Toll-like receptorsin inflammation 6

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Toll-like receptors: signaling
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Rakoff-Nahoum, S., and Medzhitov, R. (2009) Toll-like receptors and cancer. Nat RevCancer 9, 57-63
TLR8
TLR10
Recognize molecular patterns
derived from:
• Microbes
• Endogenous molecules
Either:
• Directly
• Indirectly (with the help of
accessory molecules)
Both at:
• Plasma membrane
• Intracellular compartments
Signal through:
• MYD88
• TRIF
TLR activation leads to:
• Regulation of innate immune
response
• Regulation of adaptive
immune responses
• Inflammation
• Tissue repair

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Ligands of TLRs – host factors can be ligands too!
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Chen, K., and Huang, J., et al. (2007) Toll-like receptors in inflammation, infection and cancer. Int. Immunopharmacology, 7, 1271

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Toll-like receptors: expression
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Ospelt, C., and Gay, S. (2010) TLRs and chronic inflammation. Int. J. Biochem. Cell Biol. 42, 495
TLRs are expressedin a wide variety of cells
TLR Expression
TLR1
Monocytes, macrophages, B cells, T cells, DCs, PMN, NK cells, non-
immune cells (fibroblasts, astrocytes, epithelial cells, keratinocytes)
TLR2
Monocytes, macrophages, DCs, PMN, non-immune cells (fibroblasts,
astrocytes, epithelial cells, keratinocytes)
TLR3
DCs, macrophages, mast cells, NK cells, non-immune cells
(fibroblasts, astrocytes, epithelial cells, keratinocytes)
TLR4
Monocytes, macrophages, DCs, PMN, non-immune cells (fibroblasts,
astrocytes, epithelial cells, keratinocytes)
TLR5
Monocytes, macrophages, T cells, DCs, PMN, non-immune cells
(fibroblasts, astrocytes, epithelial cells, keratinocytes)
TLR6
Monocytes, macrophages, B cells, T cells, DCs, PMN, NK cells, non-
immune cells (fibroblasts, astrocytes, epithelial cells, keratinocytes)
TLR7 B cells, plasmacytoid DCs
TLR8 Monocytes, myeloid DCs
TLR9 B cells, plasmacytoid DCs, GI epithelial cells, keratinocytes
TLR10 B cells, plasmacytoid DCs

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Toll-like receptor signaling: transcriptional control
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Post-translational activation
of transcription factors after
TLR stimulation
Transcription factors are induced
during the primary response
Transcription factors are
cell-specific
Induction of three classes of transcription factors
Medzhitov, R., and Horng, T. (2009) Transcriptional control of the inflammatory response. Nat. Rev. Immunol. 9, 692

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TLRs in inflammation
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Activation of TLRs leads to a complex,
module-specific, inflammatory transcriptional
response based on the pathways activated
Medzhitov, R., and Horng, T. (2009) Transcriptional control of the inflammatory response. Nat. Rev. Immunol. 9, 692

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Parker, L.C., Prince, L.R., and Sabroe, I. (2007) Translational mini-review series on Toll-like receptors:
networks regulated by Toll-like receptors mediate innate and adaptive immunity. Clin. Exp. Immunol. 147, 199
TLRs in inflammation: linking innate and adaptive immuneresponses
TLRs form a bridge between innate and adaptive immune responses

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TLRs are associated with diverse diseases
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Inflammation Allergy Brain Ischemia
Cancer Diabetes Mellitus Myocarditis
Infection Metastasis Pneumonia
Injury Insulin Resistance Septic Shock
Mutation Tuberculosis Brain Injury
Atherothrombosis Rheumatoid Arthritis Pain
Breast Tumor Prostate Cancer Lung Cancer
Anoxia Stroke Systemic Lupus Erythematosus
Septicemia Cirrhosis Necrosis
Death Myocardial Infarction Thrombosis
Ischemia Periodontitis
Autoimmunity Large Cell Lymphoma
Cardiac Failure Squamous Cell Carcinoma
Autoimmune Disease Infectious Disease
Arthritis Bacterial Infection
Reperfusion Injury Lupus
Hepatobiliary Disease/Toxicity Melanoma
Asthma Acne Vulgaris
Colitis Type 2 Diabetes Mellitus
Type 1 Diabetes Mellitus Stomach Cancer
https://www.qiagen.com/shop/genes-and-pathways/knowledgehub/pubmed-publications/

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Research solutions at QIAGEN
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miRNA PCR ArraysGene expression lncRNA PCR Arrays
Pathway-focused approach
lncRNA PCR Arrays

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TLR signaling RT2 Profiler PCR Array: an application example
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TLR8 HIV
?
How does TLR8 signaling lead to latent
HIV production from myeloid-monocytic
cells and from CD4 T-cells?
www.jimmunol.org/content/186/7/4314.long
Myeloid-
monocytic
cells
CD4
T cells
HIV

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QIAGEN tools for studying TLRs in inflammation
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RT2 Profiler PCRArrays & Assays
Sample
Isolation &
Preparation
Assay
Set-up & Analysis
Culture cells with
cells isolated from
blood samples of
6 HIV+ patients
RT2 TLR Signaling PCR Array

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QIAGEN tools for studying TLRs in inflammation
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• Profile 84 different genes
using one array
• Appropriate controls for
◦ data normalization
◦ sample quality
◦ reaction performance
• Allows you to focus on
your questions and papers
Gene expressionprofiling - RT2 Profiler PCRArrays & Assays

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Toll-like Receptor Signaling PCR Array
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Toll-Like Receptor Signaling Pathway RT2 Profiler PCR Array
https://www.qiagen.com/usearch/rt2-profiler-pcr-arrays?catno=PAHS-018Z#geneglobe
Guaranteed system
• Relevance
• Sensitivity
• Reproducibility
• Specificity
• Uniform PCR
amplification efficiency

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TLR8
Toll-like receptors: signaling
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Foster, S.L., and Medzhitov, R. (2009) Gene-specific control of the TLR-induced inflammatory response. Clin. Immunol. 130(1), 7

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Toll-like receptors: signaling
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Using RT2 Profiler TLR PCRArrays to analyze pathway activity

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TLR8
Toll-like receptors: signaling
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Foster, S.L., and Medzhitov, R. (2009) Gene-specific control of the TLR-induced inflammatory response. Clin. Immunol. 130(1), 7
Further
activation
of latent
HIV in CD4
T-cells
TNF-alpha

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QIAGEN tools for studying TLRs in inflammation
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TLR signaling pathway Antibacterial responses
NFkB signaling pathway Antiviral responses
NFkB signaling targets Antifungal responses
MAPK signaling pathway Inflammatory responses and autoimmunity
PI3K-Akt signaling pathway Innate and adaptive immune responses
Th1 & Th2 responses Cytokines & chemokines
Interferons & receptors Over 170 other pathways
Inflammasomes 13 species
Available for: human, mouse, rat, monkey, dog, zebrafish, rabbit, pig, fruit fly,
chicken, horse, cow and CHO cells
RT2 Profiler PCRArrays & Assays

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Research solutions at QIAGEN
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miRNA PCR ArrayslncRNA PCR ArraysGene expression
Pathway-focused approach
lncRNA PCR Arrays

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lncRNAs: new players in human diseases
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• lncRNAs are involved in gene
regulatory networks that govern
host-pathogeninteractions
• Toll-like receptors induce the
expression of lncRNAs
• lncRNA-Cox2 mediates both the
activation and repression of
distinct classes of immune genes
• lncRNAs are involved in the
control of NFkB signaling and
inflammation
• PACER controls Cox-2
expression by binding the
repressive NF-kB subunit p50
Carpenter, S., et al. (2013) A Long NoncodingRNA Mediates Both Activation and Repression of Immune Response Genes. Science. 341, 789
Krawczyk, M., and Emerson, B.M. (2014) p50-Associated COX2 Extragenic RNA (PACER) activates COX-2 gene expression by occluding
repressive NF-κB complexes. Elife. 3:e01776
• lncRNAs play important roles in a wide range of biological processes
• Dysregulated lncRNAs are involved in many complex human diseases.

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Molecular functions of lncRNAs
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Martin, L., Chang, H.Y. (2012) Uncovering the role of genomic "dark matter" in human disease. J Clin. Invest. 122, 1589
lncRNAs are a novel class of RNAs, larger than 200 nucleotides

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QIAGEN tools for studying TLRs in inflammation
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• lncRNA databases
◦ In-house databases at QIAGEN GeneGlobe
◦ Provide more than 40,000 human and 27,000 mouse lncRNAassays
• RT2 lncRNA Assays
◦ Laboratory-verifiedfor optimal qPCR performance– high specificity,
amplification efficiency and sensitivity
• RT2 lncRNA qPCR Arrays
◦ Pathway or disease-relevant lncRNAassays
◦ RT2 lncRNAInflammatory Response and Autoimmunity Array and RT2
lncFinder PCR Array (Human & Mouse)
• Custom options
◦ Flexible custom designs from the lncRNAand qPCR databases to
profile mRNAs and lncRNAs simultaneously
• lncRNA isolation
◦ miRNeasy and exoRNeasy kits
• Free on-line data analysis tool
http://www.qiagen.com/us/landing-pages/lncrna/
RT2 lncRNA PCR Arrays and Assays

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Experiment design: discover lncRNAs in your total RNA samples
Toll-like receptors in inflammation 27
2. cDNAsynthesis: convert RNAto cDNA.
3. Pre-amplification with RT2 PreAMP cDNAkit.
1. Total RNAisolation.
4.
5.
Samples: control and treated
• Total RNA isolation: isolate total RNA with miRNeasy Kits
• cDNA synthesis: convert total RNA to cDNA using RT2 First
Strand Kit
• Pre-amplification: Pre-amplify the target lncRNAs with
pre-amplification RT2 PreAMP Primer mixes (optional)
• Run PCR: detect lncRNAs with RT-PCR
◦ Human RT2
lncRNA Cancer PathwayFinder PCR Array
◦ Master mix: RT2
SYBR®
Green qPCR Mastermix
◦ qPCR cyclers
• Free data analysis: GeneGlobe Data Analysis Center
RT2 lncRNA PCR Array Inflammatory Response & Autoimmunity
84 lncRNAs involved in:
• Acute-Phase Response
• Autoimmunity
• Humoral Immune Response
• Inflammatory Response
• Innate & Adaptive Immunity
• Other Inflammatory Response & Autoimmunity

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Regulation of TLR signaling through miRNAs
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Quinn, S.R., and O’Neil, L.A. (2011) A trio of microRNAs that control Toll-like receptor signalling. Int. Immunol. 23, 421
miRNAs can be
• upregulated by TLR signaling AND
• feedback and regulate TLR pathway responses
miR-155 Targets
miR-146a Targets
miR-21 Targets
let-7 Targets
miR-1 and miR-206 Targets
miR-153 Targets

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Research solutions at QIAGEN
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lncRNA PCR Arrays miRNA PCR ArraysGene expression
Pathway-focused approach
lncRNA PCR Arrays

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miRNA biogenesis – like a gene or part of exon/intron
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• Transcribed by RNA polymerase II as long
primary transcripts (pri-miRNAs), which may
contain more than one miRNA
• In the nucleus, pri-miRNAs are processed to
hairpin-like pre-miRNAs by RNAse III-like
enzyme Drosha
• Pre-miRNAs are then exported to the cytosol
by Exportin 5
• In the cytosol, RNAse III-like Dicer processes
pre-miRNAs into mature miRNAs
• Mature miRNAs are incorporated into RISC
• miRNAs with high homology to target
mRNAs lead to mRNA cleavage
• miRNAs with imperfect base pairing to target
mRNAs lead to translational repression
and/or mRNA degradation

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miRNA array application: miR-146 in negative feedback loops
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Application example:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1567904/

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Expression of miRNAs after activation of TLRs
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Microarray results are validated
by RT-PCR
Induce THP-1 cells (human acute monocytic leukemia) with LPS
Microarray analyses

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Expression of miR-146a is NF-κB-dependent
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Mutations in NF-κB binding site
in the genomic loci of miR-146a
Taganov, K.D., Boldin, M.P., Chang, K.J., and Baltimore, D. (2006) NF-kappaB-dependent induction of
microRNA miR-146, an inhibitor targeted to signalingproteins of innate immune responses. PNAS 103, 12481

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miR-146 targets mediators downstream of TLR
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Transfect cells with TRAF6 or IRAK1 reporter constructs
Transfect cells with miRNAs
Read luciferase activity
http://www.ncbi.nlm.nih.gov/pubmed/16885212
miR-146a Targets PCRArrays
Taganov, K.D., Boldin, M.P., Chang, K.J., and Baltimore, D. (2006) NF-kappaB-dependent induction of
microRNA miR-146, an inhibitor targeted to signalingproteins of innate immune responses. PNAS 103, 12481

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microRNAs associatedwith TLR signaling
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miR-146a miR-146 miR-132 let-7e miR-143
miR-155 miR-145 miR-29c miR-181a miR-212
let-7i miR-210 miR-107 miR-302b miR-1915
miR-34c miR-27b miR-31 miR-16 miR-34a
miR-147 miR-574 miR-1225 miR-187 miR-466l
miR-125a miR-92a miR-146b miR-128a miR-149
miR-21 miR-202 miR-200c miR-200a miR-7
miR-26a miR-29b miR-200b miR-221 miR-23a
miR-125b miR-301a miR-126 miR-10a miR-223
let-7b miR-9 miR-17 miR-142 miR-150
https://www.qiagen.com/shop/genes-and-pathways/knowledgehub/pubmed-publications/

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QIAGEN tools for studying TLRs in inflammation
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miScript miRNA PCR Arrays
• 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 (>20 arrays)
◦ miFinder
◦ Inflammatory Response
◦ Autoimmunity
◦ T-Cell & B-Cell Activation
◦ Immunopathology
◦ Cell Differentiation & Development
◦ Serum & Plasma
◦ Apoptosis
miScript PreAMP Kit
• Optional step for small or precious samples
• Full miRNome profiling from as little as 1 ng RNA
• Pre-formatted, single-use PCR arrays with wet-lab verified assays
• miRNA function can be analyzed using microRNAmimics, inhibitors and target protectors

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QIAGEN tools for studying TLRs in inflammation
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miScript miRNA PCRArray workflow
1. Isolate total RNA with miRNeasy kits
2. Perform reverse-transcription
3. Prepare PCR pre-mix
4. Load arrays & perform real-time PCR
5. Analyze Data
1 hour
2 minutes
2 hours
5 minutes

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QIAGEN tools for studying TLRs in inflammation
Toll-like receptorsin inflammation 38
• Profile 84 different miRNAs
using one array
• Appropriate controls for
◦ data normalization
◦ sample quality
◦ reaction performance
• Allows you to focus on
your questions and papers
miScript miRNA PCRArrays
http://www.qiagen.com/search/miscript-mirna-pcr-arrays

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QIAGEN TLR signaling research portal
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https://www.qiagen.com/shop/genes-and-pathways/complete-biology-list/toll-like-receptor-signaling/

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QIAGEN Toll-Like Receptor Signaling ResearchSolutions
Solutions & Technologies
• RT2 Profiler PCR Array
• RT2 lncRNAPCR Array
• miScript miRNA PCR Array
• EpiTect MethylationArrays
• EpiTect Chromatin IP
• siRNA and shRNA
• Mutation analysis
• Multi-Analyte ELISArry Kits
• Cignal Reporter Assays
https://www.qiagen.com/shop/genes-and-pathways/complete-biology-list/toll-like-receptor-signaling/

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Thank you for attending today’s webinar!
Contact QIAGEN
Call: 1-800-426-8157
Email: BRCSupport@qiagen.com
qiawebinars@qiagen.com
Questions?
41
Samuel J. Rulli, Ph.D.
Samuel.Rulli@qiagen.com