DTU - January 2007 - Hanne Jarmer Introduction to DNA microarrays DTU - January 2007 - Hanne Jarmer
Microarrays - The Concept Measure the level of transcript from a very large number of genes in one go RNA CELL
Why? RNA
gene specific DNA probes How? gene mRNA gene specific DNA probes labeled target
Microarrays - The Technologies Stanford-type Microarrays High-density
Stanford-type Microarrays
Stanford-type Microarrays Coating glass slides Deposition of probes Post-processing Hybridization
Spotting - Mechanical deposition of probes
16-pin microarrayer
Microarrayer
Stanford microarrays SAMPLE CONTROL mRNA cDNA Cy3-cDNA Cy5-cDNA
Affymetrix GeneChip® oligonucleotide array • 11 to 20 oligonucleotide probes for each gene On-chip synthesis of 25 mers ~20,000 genes per chip good quality data 280-500 K features to play with
Photolithography Mask #2 Mask #1 in situ synthesis T A Mask #2 Mask #1 T A Spacers bound to surface with photolabile protection groups
Sample Preparation - Eberwine RNA 42 C 2 h ssDNA + Reverse Transcriptase 70 C 10 min T7 + RNase H + Polymerase 16 C 2 h T7 pol clean up dsDNA dsDNA 37 C 6 h + Biotin-labeled nucleotides aRNA
Detection of Biotin (Affymetrix) Streptavidin Phycoerythrim = SAPE ( ) anti-SAPE IgG biotinylated anti-anti IgG
The Affymetrix GeneChip® A gene is represented like this: PM MM - Perfect Match (PM) - MisMatch (MM) PM: CGATCAATTGCACTATGTCATTTCT MM: CGATCAATTGCAGTATGTCATTTCT
NimbleGen 385,000 to 2.1 mill features Long probes (up to 70 nt) Service: labelling scanning image analysis
Photolithography - Micromirrors
Analysis of Data Normalization: Linear or non-linear
Is it worth it? Known positives versus the total number of significantly affected genes at 5 different cutoffs in the TnrA experiment Number of known positives Qspline normalization Linear normalization Number of significantly affected genes
Analysis of Data Normalization: Linear or non-linear Statistical test: student’s t-test ANalysis Of VAriance (ANOVA) Analysis: Principal Component Analysis (PCA) Clustering and visualization
Tiling arrays Tiling arrays are used for determation of genes, ncRNAs, TF-binding sites, ...
Regulatory T Cells and Allergy Hanne Jarmer (CBS)
Type I Hypersensitivity A normal reaction against parasites Typical allergens: - pollen - penicillin - nuts - milk - bee venom - mold spores
Early Last Century Poul Portier and Charles Richet observed: Portuguese Man of War “Hmm..., maybe we can make a vaccine?”
Early last century
Immediate Hypersensitivity Richet got the Nobel Prize Early Last Century 1. time: No reaction 2. time: Got sick died Immediate Hypersensitivity anaphylaxis Richet got the Nobel Prize
The Mechanism
The Regulation Several factors are important: - genetics presentation (concentration/mode) - TH1, TH2 and TReg
The Regulation TC TH1 TH2 TReg The Naive T cell will differentiate into different subsets induced by different cytokines Naive T cell Cytokines TH1 TH2 TReg TC
The Regulation Naive T cell IL-12 IL-4 IL-10 ? TH1 TReg TH2
CD4+ T cells - The normal state - Keeping the balance TH1 TH2 TReg
The allergic state - Out of balance TReg TH1 TH2
Three types of TReg cells CD25 TReg1 TReg2 TReg3 IL-10 TGF- CD25 = IL-2 receptor (IL2RA)
CD25+ TReg cells Low proliferative capacity Immunoregulatory properties T cell homeostasis Prevents autoimmunity Antigen specific, cell-cell adhesion Induction of tolerance ... but how? Potential use in Immuno Specific Therapy (SIT) - graft tolerance ... or the other way around ... in vaccines or cancer treatment
Microarray Project The goal: Investigate the mechanism find cure for allergy or an effective SIT The plan: Find the responsible genes by the use of carefully designed microarray experiments
The Microarray Experiments Blood from 5 normal and 5 allergic patients normal allergic normal CD4+CD25+ CD4+ allergic 20 microarrays
The Microarray Experiments 20 microarrays
1 2-way ANOVA 2 Normal Allergic Normal Allergic CD4+CD25+ CD4+CD25+
2-way ANOVA
The Regulatory T cell signal Natural Treg cells Nature Immunology 6, 345 - 352 (2005) Naturally arising Foxp3-expressing CD25+CD4+ regulatory T cells in immunological tolerance to self and non-self by Shimon Sakaguch
Fewer active TReg cells No tolerance Maybe ... CTLA-4 B7 X APC TReg (TCR MHC-II) Allergic TReg: Less X Fewer active TReg cells No tolerance
Thanks to Liu Anting, PhD from ALK ALK-abello Kristine Dahlin and Thomas Jensen