normal carcinome inflammation - Life-Science Lab...pancreas EU Profiling Pancreatic Cancer Pancreatic Cancer vs. Chronic Pancreatitis carcinome normal inflammation Correspondence Analysis;
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carcinomenormal
inflammation
DKFZ Genomics Activities
Basic Level of Functional Analysis
DNA RNA ProteinDNA RNA Protein
Enzymatic activity
DNA RNA ProteinDNA RNA Protein
Structure
Organisation
Splicing
RNAi
Localisation
Combination
ModificationInteraction
Copy numberStructure
DegradationComposition
Transcription ExpressionMethylation
SNPs
Basic Level of Functional Analysis
DNA RNA ProteinDNA RNA Protein
Structure
Organisation
Splicing
RNAi
Localisation
Combination
ModificationInteraction
Copy numberStructure
DegradationComposition
Transcription ExpressionMethylation
SNPs
Basic Level of Functional Analysis
Enzymatic activity
Hoheisel, J.D. (2006) Nature Rev. Genet., in press.
Microarrays
Robotic Production
Drosophila Microarray
Hild et al. (2003)Genome Biol. 5, R3.Boutros et al. (2004)Science 303, 382-385.Hollich et al. (2004)Biotechniques 37, 282-284.
Drosophila Microarray
In-House In Situ Synthesis of Oligomer Chips
Oligomer Synthesis Within the Channels
Features (Microscopic View)
Mask-Free Control of Oligonucleotide Synthesis
1. Molecules bound to glass surface
2. Cleavage of protection groups at illuminated spots
3. Condensation of amidites results inoligomerelongation
Light
Amidites
Digital micromirror system
Photolabileprotection group
1. 2. 3.
Oligomer Synthesis Within the Channels
Epigenetic Measurements
methC methC CC U T
bisulfite PCR
Typinganalysis
healthyhealthy
tumourtumour
Functional Genome AnalysisEpigenetics
DFG
5'...-GA
GC
AG
TGG
AC
G...-3'
5'...-GA
GC
AG
CG
GA
CG
...-3'
sequence polymorphism
type 1
type 2
type 1type 2
JHGCCAAGCTGGAGCCGAGCTG
GAGCCCAGCTG
GAGCCTAGCTG
AAGCTATACGT
AAGCTGTACGT
AAGCTCTACGT
AAGCTTTACGT
CGTCGAATGCT
CGTCGGATGCT
CGTCGCATGCT
CGTCGTATGCT
CCAGGAGTCTC
CCAGGGGTCTC
CCAGGCGTCTC
CCAGGTGTCTC
ab
c d
ab
c d
TypingS
ingle Nucleotide
Polym
orphisms
Oligomers for methylated DNAOligomers for unmethylated DNA
PCR-product made from unmethylated TIMP3 promoter DNA
Mund et al. (2005) Nucleic Acids Res. 33, e73.
Detection by Mismatch Discrimination
Transcriptional Profiling
cDNA microarrayscanned image
sample 1 sample 2
gene 1 gene 2 gene 3
RNA
ManMouseDrosophila melanogasterArabidopsis thalianaSaccharomyces cerevisiaeHydractinia echinata
Bacillus subtilisTrypanosoma bruceiNeurospora crassaPseudomonas putidaRhizobium meliloti
EU
EU
EU
Transcriptional Profiling Projects
pancreas
EU
Profiling Pancreatic Cancer
Pancreatic Cancer vs.
Chronic Pancreatitis
carcinomenormal
inflammation
Correspondence Analysis; Fellenberg et al. (2001) PNAS 98, 10781
patient
differentiallytranscribed gene
M-CHiPSData Processing Software
Target Selection
Buchholz et al. (2005) Clin. Cancer Res. 11, 8048-8054.
Target Selection
carcinomenormal
M-CHiPSData Processing Software
Correspondence Analysis; Fellenberg et al. (2001) PNAS 98, 10781
differences between individualsdifferences between individuals
inflammation-specific genesinflammation-specific genestumour-specific genestumour-specific genes
inflammation
Buchholz et al. (2005) Clin. Cancer Res. 11, 8048-8054.
M-CHiPSData Processing Software
Transcript Profiling Social Behaviour
healthycontrol
� MCA� MCAC
� SCA� IPMT
carcinomenormal
inflammation
normal tissue
ductal adenocarcinoma
cystic tumours
case tumour
cystictumours
Complex Antigen-Antibody InteractionsAntibody Microarrays
illustration
Kusnezow & Hoheisel (2002) BioTechniques 33 (suppl.), 14-23.Kusnezow et al. (2003) Proteomics 3, 254-264.Kusnezow & Hoheisel (2003) J. Mol. Recognit. 16, 165-176.Kusnezow et al. (2004) Protein Microarrays (Schena, M., ed.), 247-284.Kusnezow et al. (2005) Handbook of Immunohistochemistry
(Hayat, M.A., ed.), 23-37.
real experiment
EU
Kusnezow et al. (2006) Expert Rev. Mol. Diagn. 6, 111-124Kusnezow et al. (2006) Proteomics, in press.
Selection of Antibodies
TranscriptionalProfiling
Expression Profiling
• DNA sequences of 900 genes/ESTsexhibiting differential transcription
• Peptide selection from sequences according to 12 selection criteria
• Peptide synthesis
• Immunisation of rabbits
• Affinity purification of polyclonal antibodies
• Characterisation by in situ analyses
Antibody Microarrays
ProteinBinder Consortium
EU
Serum Sample AnalysisAntibody Microarrays
EU
Analysing Pancreatic Cancer
Transcript Profiling
pancreas
EU
Epigenetic Analysis
Protein Expression
www.dkfz.de/funct_genome
Functional Genome Analysis, DKFZ, Heidelberg
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