1. InflammaTools: Complete research tools to study
Inflammation regulation
Raed Samara, Ph.D.
R&D Scientist
Sample & Assay Technologies

2. Biology-focused solutions for pathway analysis
• SABiosciences is now a
Company
-2-
Sample & Assay Technologies

3. Introduction: Inflammation
̣
Definition: a protective tissue response to tissue damage or microbes, which serves to
destroy, dilute, or wall off both the injurious agent and the injured tissues.
Epigenetic
Changes
Microbes/Infection
Tissue Damage
Acute Inflammation
Infection Clearance
Tissue Homeostasis
mRNA
Changes
Cytokines & Chemokines
Signaling Pathways
Immune system composition
Chronic Inflammation
Pre-cancer & Cancer
Chronic Inflammatory Diseases
-3-
Sample & Assay Technologies

4. Inflammation experimental design & QIAGEN
Gene Expression
RT-PCR
Epigenetics
miRNA
DNA methylation
Histone modifications
Functional Studies
Reporter assays
siRNA/shRNA
Protein Expression
Cytokine levels
̣
̣
̣
̣
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Sample & Assay Technologies

5. Basic principles of qRT-PCR: Overview
Real-Time PCR
Amplify and simultaneously quantify target DNA
Reverse Transcription Real-Time PCR
Amplify and simultaneously quantify mRNA
For more information and webinars on real-time PCR, visit:
www.sabiosciences.com/seminarlist.php
-5-
Sample & Assay Technologies

6. Experimental overview: Gene expression
analysis
Isolate RNA
Stimulate Cells
RT-PCR Arrays or Assays
Isolate DNA
Data Analysis
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Sample & Assay Technologies

7. Gene expression in Inflammation
̣
Definition: a protective tissue response to tissue damage or microbes, which serves to
destroy, dilute, or wall off both the injurious agent and the injured tissues.
Epigenetic
Changes
Microbes/Infection
Tissue Damage
Acute Inflammation
Infection Clearance
Tissue Homeostasis
mRNA
Changes
Cytokines & Chemokines
Signaling Pathways
Immune system composition
Chronic Inflammation
Pre-cancer & Cancer
Chronic Inflammatory Diseases
-7-
Sample & Assay Technologies

8. ̣
Inflammation studies: Gene expression
The transcription factor cMyb is upregulated in naïve CD4 T cells.
-8-
Sample & Assay Technologies

9. ̣
Inflammation studies: Gene expression
The transcription factor cMyb is upregulated in naïve CD4 T cells.
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Sample & Assay Technologies

10. ̣
Inflammation studies: Gene expression
The transcription factor cMyb is upregulated in naïve CD4 T cells. What is the effect of cMyb
on naïve CD4 T cell maturation (to Th1, Th2, or Th3)?
- 10 -
Sample & Assay Technologies

11. Inflammation studies: Gene expression
The transcription factor cMyb is upregulated in naïve CD4 T cells. What is the effect of cMyb
on naïve CD4 T cell maturation (to Th1, Th2, or Th3)?
̣
Experiment: Knockdown cMyb in human peripheral CD4+ T cells. Examine gene changes
using the Human Th1-Th2-Th3 RT2 Profiler PCR Array (PAHS-034).
̣
- 11 -
Sample & Assay Technologies

12. Inflammation studies: Gene expression
The transcription factor cMyb is upregulated in naïve CD4 T cells. What is the effect of cMyb
on naïve CD4 T cell maturation (to Th1, Th2, or Th3)?
̣
Experiment: Knockdown cMyb in human peripheral CD4+ T cells. Examine gene changes
using the Human Th1-Th2-Th3 RT2 Profiler PCR Array (PAHS-034).
̣
Results:
̣
- 12 -
Sample & Assay Technologies

13. Inflammation studies: Gene expression
The transcription factor cMyb is upregulated in naïve CD4 T cells. What is the effect of cMyb
on naïve CD4 T cell maturation (to Th1, Th2, or Th3)?
̣
Experiment: Knockdown cMyb in human peripheral CD4+ T cells. Examine gene changes
using the Human Th1-Th2-Th3 RT2 Profiler PCR Array (PAHS-034).
̣
Results:
̣
Conclusions: Knockdown of cMyb in human peripheral CD4+ T cells decreased the
expression of Th2 cytokine genes, and negatively affected Th2 cell maturation in primary
human peripheral blood T cells.
̣
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Sample & Assay Technologies

14. Gene expression in Inflammation
̣
Definition: a protective tissue response to tissue damage or microbes, which serves to
destroy, dilute, or wall off both the injurious agent and the injured tissues.
Epigenetic
Changes
Microbes/Infection
Tissue Damage
Acute Inflammation
Infection Clearance
Tissue Homeostasis
mRNA
Changes
Cytokines & Chemokines
Signaling Pathways
Immune system composition
Chronic Inflammation
Pre-cancer & Cancer
Chronic Inflammatory Diseases
- 14 -
Sample & Assay Technologies

15. ̣
Inflammation studies: Gene expression
Limited material
Mucin depleted foci (MDF) are precancerous lesions of the colon that show signs of
inflammation.
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Sample & Assay Technologies

16. ̣
Inflammation studies: Gene expression
Limited material
Mucin depleted foci (MDF) are precancerous lesions of the colon that show signs of
inflammation.
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Sample & Assay Technologies

17. ̣
Inflammation studies: Gene expression
Limited material
Mucin depleted foci (MDF) are precancerous lesions of the colon that show signs of
inflammation. What roles do TLRs play in MDF?
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Sample & Assay Technologies

18. Inflammation studies: Gene expression
Limited material
Mucin depleted foci (MDF) are precancerous lesions of the colon that show signs of
inflammation. What roles do TLRs play in MDF?
̣
Experiment: Treat mice with DMH for 15 weeks to induce MDF. Collect RNA from MDF and
normal colon. Convert mRNA to cDNA. Pre-amplify cDNA with the Mouse TLR PreAMP
primer mixes (PBM-0018). Analyze differential gene expression with the Mouse TLR RT2
Profiler PCR Arrays (PAMM-018)
̣
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Sample & Assay Technologies

19. Inflammation studies: Gene expression
Limited material
Mucin depleted foci (MDF) are precancerous lesions of the colon that show signs of
inflammation. What roles do TLRs play in MDF?
̣
Experiment: Treat mice with DMH for 15 weeks to induce MDF. Collect RNA from MDF and
normal colon. Pre-amplify cDNA with the Mouse TLR PreAMP primer mixes (PBM-0018).
Analyze differential gene expression with the Mouse TLR RT2 Profiler PCR Arrays (PAMM018)
̣
Results:
̣
- 19 -
Sample & Assay Technologies

20. Inflammation studies: Gene expression
Limited material
Mucin depleted foci (MDF) are precancerous lesions of the colon that show signs of
inflammation. What roles do TLRs play in MDF?
̣
Experiment: Treat mice with DMH for 15 weeks to induce MDF. Collect RNA from MDF and
normal colon. Pre-amplify cDNA with the Mouse TLR PreAMP primer mixes (PBM-0018).
Analyze differential gene expression with the Mouse TLR RT2 Profiler PCR Arrays (PAMM018)
̣
Results:
̣
Conclusion: MDF induces TLR2 gene expression in MDF compared to normal colon,
suggesting a link between TLR2 and MDF.
̣
- 20 -
Sample & Assay Technologies

21. Inflammation experimental design & QIAGEN
Gene Expression
RT-PCR
Epigenetics
miRNA
DNA methylation
Histone modifications
Functional Studies
Reporter assays
siRNA/shRNA
Protein Expression
Cytokine levels
̣
̣
̣
̣
- 21 -
Sample & Assay Technologies

22. Epigenetics: Overview
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
For more information and webinars on Epigenetics, visit:
www.sabiosciences.com/seminarlist.php
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Sample & Assay Technologies

23. miRNA reagents from QIAGEN
miRNeasy
Isolation
Target Identification
miRNA
Studies
Expression
miScript miRNA PCR Arrays
3’ UTR Reporters
Function
miRNA mimics & inhibitors
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Sample & Assay Technologies

24. miRNA in Inflammation
̣
Definition: a protective tissue response to tissue damage or microbes, which serves to
destroy, dilute, or wall off both the injurious agent and the injured tissues.
Microbes/Infection
Tissue Damage
Epigenetic
Changes
(miRNA)
mRNA
Changes
Acute Inflammation
Infection Clearance
Tissue Homeostasis
Cytokines & Chemokines
Signaling Pathways
Immune system composition
Chronic Inflammation
Pre-cancer & Cancer
Chronic Inflammatory Diseases
- 24 -
Sample & Assay Technologies

25. ̣
Inflammation studies: miRNA function
Resolution of inflammation is a tightly controlled mechanism. The D-series resolvins (RvD1)
are a class of players that mediate this resolution.
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Sample & Assay Technologies

26. ̣
Inflammation studies: miRNA function
Resolution of inflammation is a tightly controlled mechanism. The D-series resolvins (RvD1)
are a class of players that mediate this resolution.
FASEB J. 25, 1–17 (2011)
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Sample & Assay Technologies

27. ̣
Inflammation studies: miRNA function
Resolution of inflammation is a tightly controlled mechanism. The D-series resolvins (RvD1)
are a class of players that mediate this resolution. Can RvD1-mediated resolution happen
through miRNAs?
FASEB J. 25, 1–17 (2011)
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Sample & Assay Technologies

28. Inflammation studies: miRNA function
Resolution of inflammation is a tightly controlled mechanism. The D-series resolvins (RvD1)
are a class of players that mediate this resolution. Can RvD1-mediated resolution happen
through miRNAs?
̣
̣
Experiment: Treat mice with zymosan A to induce acute inflammation (murine peritonitis), in
the presence or absence of RvD1, for 4, 12, or 24 hours (representing onset, maximum,
and resolution phases). Collect RNA from leukocytes. Analyze miRNA expression with the
Mouse miRNome miRNA PCR Array (MIMM-216Z)
FASEB J. 25, 1–17 (2011)
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Sample & Assay Technologies

29. Inflammation studies: miRNA function
Resolution of inflammation is a tightly controlled mechanism. The D-series resolvins (RvD1)
are a class of players that mediate this resolution. Can RvD1-mediated resolution happen
through miRNAs?
̣
Experiment: Treat mice with zymosan A to induce acute inflammation (murine peritonitis), in
the presence or absence of RvD1, for 4, 12, or 24 hours (representing onset, maximum,
and resolution phases). Collect RNA from leukocytes. Analyze miRNA expression with the
Mouse miRNome miRNA PCR Array (MIMM-216Z)
̣
̣
Results:
- 29 -
Sample & Assay Technologies

30. Inflammation studies: miRNA function
Resolution of inflammation is a tightly controlled mechanism. The D-series resolvins (RvD1)
are a class of players that mediate this resolution. Can RvD1-mediated resolution happen
through miRNAs?
̣
Experiment: Treat mice with zymosan A to induce acute inflammation (murine peritonitis), in
the presence or absence of RvD1, for 4, 12, or 24 hours (representing onset, maximum,
and resolution phases). Collect RNA from leukocytes. Analyze miRNA expression with the
Mouse miRNome miRNA PCR Array (MIMM-216Z)
̣
Results:
̣
̣
Conclusion: RvD1 regulated resolution by controlling the expression of miR-219 and miR146b. These miRNAs are the first identified miRNAs in resolvin resolution circuits.
- 30 -
Sample & Assay Technologies

31. Epigenetics: Overview
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
- 31 -
Sample & Assay Technologies

32. DNA methylation in Inflammation
̣
Definition: a protective tissue response to tissue damage or microbes, which serves to
destroy, dilute, or wall off both the injurious agent and the injured tissues.
Microbes/Infection
Tissue Damage
Epigenetic
Changes
(DNA Methylation)
mRNA
Changes
Acute Inflammation
Infection Clearance
Tissue Homeostasis
Cytokines & Chemokines
Signaling Pathways
Immune system composition
Chronic Inflammation
Pre-cancer & Cancer
Chronic Inflammatory Diseases
- 32 -
Sample & Assay Technologies

33. Inflammation studies: DNA methylation
ANCA Vasculitis is characterized by microvascular inflammation caused by activated
neutrophils. These activated neutrophils aberrantly express PR3 and MPO, which are
silenced in normal individuals.
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Sample & Assay Technologies

34. Inflammation studies: DNA methylation
ANCA Vasculitis is characterized by microvascular inflammation caused by activated
neutrophils. These activated neutrophils aberrantly express PR3 and MPO, which are
silenced in normal individuals.
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Sample & Assay Technologies

35. Inflammation studies: DNA methylation
ANCA Vasculitis is characterized by microvascular inflammation caused by activated
neutrophils. These activated neutrophils aberrantly express PR3 and MPO, which are
silenced in normal individuals.
Does aberrant PR3 and MPO expression result from disrupted epigenetic silencing?
- 35 -
Sample & Assay Technologies

36. Inflammation studies: DNA methylation
ANCA Vasculitis is characterized by microvascular inflammation caused by activated
neutrophils. These activated neutrophils aberrantly express PR3 and MPO, which are
silenced in normal individuals.
Does aberrant PR3 and MPO expression result from disrupted epigenetic silencing?
Experiment: Isolate gDNA from neutrophils and treat with methylation-sensitive and
methylation-dependent restriction enzymes (EpiTect Methyl DNA Restriction Kit 335451).
Analyze DNA methylation by real-time PCR using primers specific for CpG islands in PR3
(MePH23428-1A) and MPO (MePH22382-1A).
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Sample & Assay Technologies

37. Inflammation studies: DNA methylation
ANCA Vasculitis is characterized by microvascular inflammation caused by activated
neutrophils. These activated neutrophils aberrantly express PR3 and MPO, which are
silenced in normal individuals.
Does aberrant PR3 and MPO expression result from disrupted epigenetic silencing?
Experiment: Isolate gDNA from neutrophils and treat with methylation-sensitive and
methylation-dependent restriction enzymes (EpiTect Methyl DNA Restriction Kit 335451).
Analyze DNA methylation by real-time PCR using primers specific for CpG islands in PR3
(MePH23428-1A) and MPO (MePH22382-1A).
Results:
- 37 -
Sample & Assay Technologies

38. Inflammation studies: DNA methylation
ANCA Vasculitis is characterized by microvascular inflammation caused by activated
neutrophils. These activated neutrophils aberrantly express PR3 and MPO, which are
silenced in normal individuals.
Does aberrant PR3 and MPO expression result from disrupted epigenetic silencing?
Experiment: Isolate gDNA from neutrophils and treat with methylation-sensitive and
methylation-dependent restriction enzymes (EpiTect Methyl DNA Restriction Kit 335451).
Analyze DNA methylation by real-time PCR using primers specific for CpG islands in PR3
(MePH23428-1A) and MPO (MePH22382-1A).
Results:
Conclusion: Methylation of CpG islands is a mechanism that controls the expression of MPO,
but not the expression of PR3.
The EpiTect Methyl System uses the MethylScreen™
Technology provided under license from Orion Genomics, LLC.
- 38 -
Sample & Assay Technologies

39. Epigenetics: Overview
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
- 39 -
Sample & Assay Technologies

40. Histone modification in Inflammation
̣
Definition: a protective tissue response to tissue damage or microbes, which serves to
destroy, dilute, or wall off both the injurious agent and the injured tissues.
Microbes/Infection
Tissue Damage
Epigenetic
Changes
(Histone Modifications)
mRNA
Changes
Acute Inflammation
Infection Clearance
Tissue Homeostasis
Cytokines & Chemokines
Signaling Pathways
Immune system composition
Chronic Inflammation
Pre-cancer & Cancer
Chronic Inflammatory Diseases
- 40 -
Sample & Assay Technologies

41. Inflammation studies: Histone modification
Th17 cells are a subset of CD4 T cells that differentiate from naïve T cells, secrete IL-17, and
induce massive tissue inflammation. T cell factor 1 (TCF-1) is a transcription factor that plays
an important role in T cell differentiation in the periphery.
- 41 -
Sample & Assay Technologies

42. Inflammation studies: Histone modification
Th17 cells are a subset of CD4 T cells that differentiate from naïve T cells, secrete IL-17, and
induce massive tissue inflammation. T cell factor 1 (TCF-1) is a transcription factor that plays
an important role in T cell differentiation in the periphery.
- 42 -
Sample & Assay Technologies

43. Inflammation studies: Histone modification
Th17 cells are a subset of CD4 T cells that differentiate from naïve T cells, secrete IL-17, and
induce massive tissue inflammation. T cell factor 1 (TCF-1) is a transcription factor that plays
an important role in T cell differentiation in the periphery. Does TCF-1 control the
differentiation of Th17 cells, and how?
- 43 -
Sample & Assay Technologies

44. Inflammation studies: Histone modification
Th17 cells are a subset of CD4 T cells that differentiate from naïve T cells, secrete IL-17, and
induce massive tissue inflammation. T cell factor 1 (TCF-1) is a transcription factor that plays
an important role in T cell differentiation in the periphery. Does TCF-1 control the
differentiation of Th17 cells, and how?
Experiment: Isolate naïve T cells from spleens of mice. Induce Th17 cell differentiation.
Collect chromatin and process with the EpiTect ChIP one-day kit (334471) for chromatin
immunoprecipitation with anti-histone antibodies. Detect precipitated DNA with qRT-PCR.
- 44 -
Sample & Assay Technologies

45. Inflammation studies: Histone modification
Th17 cells are a subset of CD4 T cells that differentiate from naïve T cells, secrete IL-17, and
induce massive tissue inflammation. T cell factor 1 (TCF-1) is a transcription factor that plays
an important role in T cell differentiation in the periphery. Does TCF-1 control the
differentiation of Th17 cells, and how?
Experiment: Isolate naïve T cells from spleens of mice. Induce aTh17 cell differentiation.
Collect chromatin and process with the EpiTect ChIP one-day kit for chromatin
immunoprecipitation with anti-histone antibodies. Detect precipitated DNA with qRT-PCR.
Results:
Open chromatin = increased expression
- 45 -
Sample & Assay Technologies

46. Inflammation studies: Histone modification
Th17 cells are a subset of CD4 T cells that differentiate from naïve T cells, secrete IL-17, and
induce massive tissue inflammation. T cell factor 1 (TCF-1) is a transcription factor that plays
an important role in T cell differentiation in the periphery. Does TCF-1 control the
differentiation of Th17 cells, and how?
Experiment: Isolate naïve T cells from spleens of mice. Induce Th17 cell differentiation.
Collect chromatin and process with the EpiTect ChIP one-day kit (334471) for chromatin
immunoprecipitation with anti-histone antibodies. Detect precipitated DNA with qRT-PCR.
Results:
Conclusion: TCF-1 mediates the repression of IL-17 locus during T cell development by
chromatin modifications.
- 46 -
Sample & Assay Technologies

47. Inflammation experimental design & QIAGEN
Gene Expression
RT-PCR
Epigenetics
miRNA
DNA methylation
Histone modifications
Functional Studies
Reporter assays
siRNA/shRNA
Protein Expression
Cytokine levels
̣
̣
̣
̣
- 47 -
Sample & Assay Technologies

48. Cignal Reporter Assays: Complete Solution
Reporter assays: Overview
Transcriptional Regulatory Elements (TRE), which establish the
specificity of each reporter
TATA
box
Reporter Construct
GFP/firefly luciferase
Tandem repeats of
TRE
EGFP
TF
FL
Upstream Signaling
Events
- 48 -
Sample & Assay Technologies

49. Reporter assays for Inflammation
̣
Definition: a protective tissue response to tissue damage or microbes, which serves to
destroy, dilute, or wall off both the injurious agent and the injured tissues.
Epigenetic
Changes
Microbes/Infection
Tissue Damage
Acute Inflammation
Infection Clearance
Tissue Homeostasis
mRNA
Changes
Cytokines & Chemokines
Signaling Pathways
Immune system composition
Chronic Inflammation
Pre-cancer & Cancer
Chronic Inflammatory Diseases
- 49 -
Sample & Assay Technologies

50. ̣
Inflammation studies: Reporter assay
Neisseria Meningitidis infections can be rapidly fatal due to acute inflammatory responses
that are mediated by capsular polysaccharides (CPS), but the innate immunostimulatory
activity of CPS is largely unknown.
- 50 -
Sample & Assay Technologies

51. ̣
Inflammation studies: Reporter assay
Neisseria Meningitidis infections can be rapidly fatal due to acute inflammatory responses
that are mediated by capsular polysaccharides (CPS), but the innate immunostimulatory
activity of CPS is largely unknown.
- 51 -
Sample & Assay Technologies

52. ̣
Inflammation studies: Reporter assay
Neisseria Meningitidis infections can be rapidly fatal due to acute inflammatory responses
that are mediated by capsular polysaccharides (CPS), but the innate immunostimulatory
activity of CPS is largely unknown. What signaling pathways are induced upon CPS
recognition?
- 52 -
Sample & Assay Technologies

53. Inflammation studies: Reporter assay
Neisseria Meningitidis infections can be rapidly fatal due to acute inflammatory responses
that are mediated by capsular polysaccharides (CPS), but the innate immunostimulatory
activity of CPS is largely unknown. What signaling pathways are induced upon CPS
recognition?
̣
Experiment: Reverse-transfect cells with 10 dual-luciferase reporter assays, individually, on a
reporter array (Cignal Finder 10-pathway Reporter Array; CCA-108L). Induce transfected
cells with CPS, and measure luciferase levels to determine which pathway(s) is(are) activated
by CPS
̣
- 53 -
Sample & Assay Technologies

54. Inflammation studies: Reporter assay
Neisseria Meningitidis infections can be rapidly fatal due to acute inflammatory responses
that are mediated by capsular polysaccharides (CPS), but the innate immunostimulatory
activity of CPS is largely unknown. What signaling pathways are induced upon CPS
recognition?
̣
Experiment: Reverse-transfect cells with 10 dual-luciferase reporter assays, individually, on a
reporter array (Cignal Finder 10-pathway Reporter Array; CCA-108L). Induce transfected
cells with CPS, and measure luciferase levels to determine which pathway(s) is(are) activated
by CPS
̣
Results:
̣
- 54 -
Sample & Assay Technologies

55. Inflammation studies: Reporter assay
Neisseria Meningitidis infections can be rapidly fatal due to acute inflammatory responses
that are mediated by capsular polysaccharides (CPS), but the innate immunostimulatory
activity of CPS is largely unknown. What signaling pathways are induced upon CPS
recognition?
̣
Experiment: Reverse-transfect cells with 10 dual-luciferase reporter assays, individually, on a
reporter array (Cignal Finder 10-pathway Reporter Array; CCA-108L). Induce transfected
cells with CPS, and measure luciferase levels to determine which pathway(s) is(are) activated
by CPS
̣
Results:
̣
Conclusions: NFkB is the major signaling pathway activated in response to CPS.
̣
- 55 -
Sample & Assay Technologies

56. shRNA/siRNA function
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
- 56 -
Sample & Assay Technologies

57. Gene knockdown for Inflammation
̣
Definition: a protective tissue response to tissue damage or microbes, which serves to
destroy, dilute, or wall off both the injurious agent and the injured tissues.
Epigenetic
Changes
Microbes/Infection
Tissue Damage
Acute Inflammation
Infection Clearance
Tissue Homeostasis
mRNA
Changes
Cytokines & Chemokines
Signaling Pathways
Immune system composition
Chronic Inflammation
Pre-cancer & Cancer
Chronic Inflammatory Diseases
- 57 -
Sample & Assay Technologies

58. ̣
Inflammation studies: siRNA knockdown
Resident alveolar macrophages (AM) constitute the first line of defense against invading lung
Streptococcus pneumoniae. Studies have shown that morphine-treated mice experience
increased mortality and bacterial outgrowth and dissemination.
- 58 -
Sample & Assay Technologies

59. ̣
Inflammation studies: siRNA knockdown
Resident alveolar macrophages (AM) constitute the first line of defense against invading lung
Streptococcus pneumoniae. Studies have shown that morphine-treated mice experience
increased mortality and bacterial outgrowth and dissemination.
The Journal of Immunology, 2008, 180: 3594–3600.
- 59 -
Sample & Assay Technologies

60. ̣
Inflammation studies: siRNA knockdown
Resident alveolar macrophages (AM) constitute the first line of defense against invading lung
Streptococcus pneumoniae. Studies have shown that morphine-treated mice experience
increased mortality and bacterial outgrowth and dissemination. What is the mechanism by
which morphine does this?
The Journal of Immunology, 2008, 180: 3594–3600.
- 60 -
Sample & Assay Technologies

61. Inflammation studies: siRNA knockdown
Resident alveolar macrophages (AM) constitute the first line of defense against invading lung
Streptococcus pneumoniae. Studies have shown that morphine-treated mice experience
increased mortality and bacterial outgrowth and dissemination. What is the mechanism by
which morphine does this?
̣
Experiment: MH-S cells (murine AM cells) were transfected with either a negative control
SureSilencing shRNA or a SureSilencing shRNA plasmid for mouse TLR9. Levels of MIP-2
were measured after treating transfected cells with morphine and Streptococcus pneumoniae
̣
The Journal of Immunology, 2008, 180: 3594–3600.
- 61 -
Sample & Assay Technologies

62. Inflammation studies: siRNA knockdown
Resident alveolar macrophages (AM) constitute the first line of defense against invading lung
Streptococcus pneumoniae. Studies have shown that morphine-treated mice experience
increased mortality and bacterial outgrowth and dissemination. What is the mechanism by
which morphine does this?
̣
Experiment: MH-S cells (murine AM cells) were transfected with either a negative control
SureSilencing shRNA or a SureSilencing shRNA plasmid for mouse TLR9. Levels of MIP-2
were measured after treating transfected cells with morphine and Streptococcus pneumoniae.
̣
Results:
̣
- 62 -
Sample & Assay Technologies

63. Inflammation studies: siRNA knockdown
Resident alveolar macrophages (AM) constitute the first line of defense against invading lung
Streptococcus pneumoniae. Studies have shown that morphine-treated mice experience
increased mortality and bacterial outgrowth and dissemination. What is the mechanism by
which morphine does this?
̣
Experiment: MH-S cells (murine AM cells) were transfected with either a negative control
SureSilencing shRNA or a SureSilencing shRNA plasmid for mouse TLR9. Levels of MIP-2
were measured after treating transfected cells with morphine and Streptococcus pneumoniae.
̣
Results:
̣
Conclusion: Morphine reduces levels of Streptococcus pneumoniae -induced MIP-2 from AM
cells in a TLR9-dependent manner.
̣
- 63 -
Sample & Assay Technologies

64. Inflammation experimental design & QIAGEN
Gene Expression
RT-PCR
Epigenetics
miRNA
DNA methylation
Histone modifications
Functional Studies
Reporter assays
siRNA/shRNA
Protein Expression
Cytokine levels
̣
̣
̣
̣
- 64 -
Sample & Assay Technologies

65. Chemokine expression in Inflammation
̣
Definition: a protective tissue response to tissue damage or microbes, which serves to
destroy, dilute, or wall off both the injurious agent and the injured tissues.
Epigenetic
Changes
Microbes/Infection
Tissue Damage
Acute Inflammation
Infection Clearance
Tissue Homeostasis
mRNA
Changes
Cytokines & Chemokines
Signaling Pathways
Immune system composition
Chronic Inflammation
Pre-cancer & Cancer
Chronic Inflammatory Diseases
- 65 -
Sample & Assay Technologies

66. ̣
Inflammation studies: Chemokine levels
Diabetes mellitus (DM) is a major risk factor for tuberculosis (TB) but the defect in protective
immunity responsible for this has not been defined.
- 66 -
Sample & Assay Technologies

67. ̣
Inflammation studies: Chemokine levels
Diabetes mellitus (DM) is a major risk factor for tuberculosis (TB) but the defect in protective
immunity responsible for this has not been defined.
The Journal of Immunology, 2010, 184: 6275–6282
- 67 -
Sample & Assay Technologies

68. ̣
Inflammation studies: Chemokine levels
Diabetes mellitus (DM) is a major risk factor for tuberculosis (TB) but the defect in protective
immunity responsible for this has not been defined. Could this be due to defective chemokine
secretion leading to delayed priming of adaptive immunity?
The Journal of Immunology, 2010, 184: 6275–6282
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Sample & Assay Technologies

69. Inflammation studies: Chemokine levels
Diabetes mellitus (DM) is a major risk factor for tuberculosis (TB) but the defect in protective
immunity responsible for this has not been defined. Could this be due to defective chemokine
secretion leading to delayed priming of adaptive immunity?
̣
Experiment: Lung homogenates from TB-infected Diabetic mice were tested for chemokine
levels using the Multi-Analyte ELISArray Kit.
̣
The Journal of Immunology, 2010, 184: 6275–6282
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Sample & Assay Technologies

70. Inflammation studies: Chemokine levels
Diabetes mellitus (DM) is a major risk factor for tuberculosis (TB) but the defect in protective
immunity responsible for this has not been defined. Could this be due to defective chemokine
secretion leading to delayed priming of adaptive immunity?
̣
Experiment: Lung homogenates from TB-infected Diabetic mice were tested for chemokine
levels using the ELISArray.
̣
Results:
̣
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Sample & Assay Technologies

71. Inflammation studies: Chemokine levels
Diabetes mellitus (DM) is a major risk factor for tuberculosis (TB) but the defect in protective
immunity responsible for this has not been defined. Could this be due to defective chemokine
secretion leading to delayed priming of adaptive immunity?
̣
Experiment: Lung homogenates from TB-infected Diabetic mice were tested for chemokine
levels using the Multi-Analyte ELISArray Kit.
̣
Results:
̣
Conclusion: Reduced levels of leukocyte chemoattractant factors including CCL2 and CCL5
at early timepoints post-infection could explain why diabetic mice are more prone to TB.
̣
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Sample & Assay Technologies

72. Conclusions
QIAGEN offers many methods to study
molecular and cellular mechanisms involved in
Inflammation:
Gene Expression
RT2 Profiler PCR Arrays & Assays
RT2 PreAMP Primer Mixes
Epigenetics
miScript miRNA PCR System
EpiTect Methyl qPCR Arrays
EpiTect ChIP qPCR Arrays
Functional Studies
Cignal Reporter Assays
SureSilencing shRNA Plasmid
Protein expression
ELISArray
̣
www.sabiosciences.com
̣
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Sample & Assay Technologies

73. Keep up to date: Follow Pathway focused biology on Facebook
Latest
information on
pathway and
disease
research,
resources and
demos.
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Sample & Assay Technologies

74. Thank you for attending!
Would you like to try an Inflammation-related PCR Array?
RT2 Profiler PCR Array Starter Pack
miScript PCR Array Starter Pack
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, OR
• RT2 SYBR Green Mastermix (2-Pack)
Call 1-888-503-3187 for more information
Email: support@SABiosciences.com
(2012 US and Canada only)
Webinar-related question?
QIAWEBINARS@QIAGEN.COM
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Sample & Assay Technologies