Mikročipová technologie a její význam v patologii Mgr. Jan Bouchal, Ph.D. Ústav klinické a molekulární patologie LF UP
Přehled Projekt HUGO 2001 (Human genome organization) První genomická vlna Expresní microarrays SNP (single nucleotide polymorphism), CGH arrays Druhá genomická vlna Genome-scale RNAi Next generation sequencing 2005/6 World class projects 1000 genomes ENCODE 2012 (Encyclopedia of DNA Elements)
Projekt HUGO - ENCODE - 30-40 tisíc genů (jen 2x více než háďátko nebo octomilka) -pouze 1,1-1,4% celého genomu jsou sekvence překládané do proteinů, 28% je přepisováno do RNA, 50% genomu představují repetitivní sekvence -SNP (single nucleotide polymorfism)
Osnova Úvod Microarrays Mikrodisekce Amplifikace mrna Hybridizace Analýza dat
Metody založené na hybridizaci Southern/Nothern analýza ISH (in situ hybridizace) FISH (fluorescenční ISH) CGH komparativní genomická hybridizace DNA čipy
CGH - Comparative Genomic Hybridization Nebo matrice s jednotlivými geny, tzv. CGH microarrays
Dual channel (obvykle tzv. cdna) expression microarrays - dva vzorky jsou analyzovány na jednom čipu - tyto vzorky už nejde počítačově oddělit a srovnat s jinými typy vzorků (různé časové intervaly, chemické působení apod.)
One channel (obvykle tzv. oligonucleotide) expression microarrays
Photolitography
Affymetrix Arrays and Photolitography
Tissue microarrays Výhodou je především šetření protilátek i laboratorní práce. Hlavní nevýhodou je analýza pouze části původní tkáně (nereprezentativnost výběru při konstrukci tissue microarray)
Protein microarrays -Large scale analysis of functional proteins -Identification of protein-protein interactions, proteinphospholipid interactions, small molecule targets, and substrates of proteins kinases Hall et al.: Protein Microarray Technology. Mech Ageing Dev. 2007; 128(1): 161
Protein microarrays cell free synthesis Chandra et Srivastava: Cell-free synthesis-based protein microarrays and their applications. Proteomics 2010; 10: 717-30.
Význam selekce jednotlivých buněčných populací Gen 1 proteoglycan Gen 2 CD53 antigen Gen 3 podjednotka proteasomu Taylor et al. Cytometry Part A 2004, 59A: 254-261
LCM, Laser Capture Microdissection (např. systém Arcturus) LMPC, Laser Microdissection and Pressure Catapulting (systém PALM)
Before LCM After LCM ILC IDC ILC IDC Duct Lobule Duct Lobule
Lineární amplifikace RNA 1000 buněk ~ cca 50 ng RNA Pro hybridizaci na microarray je třeba cca 10 ug
- normalizace čipů mezi sebou - statistické vyhodnocení rozdílně exprimovaných genů - interpretace výsledků (funkce genů, signální a metabolické dráhy, )
Data analysis Differentially expressed genes between cell types were identified using the GCOS (Gene Chip Operating Software) change algorithm and Rank Products (RP) following RMA (Robust Multiarray Analysis). Comparison Number of differentially expressed probe sets by: GCOS RP both methods Tduc vs D 1082 624 346 Tduc vs L 816 364 188 Tlob vs D 1023 350 201 Tlob vs L 983 344 208 D vs L 367 501 82 Tlob vs Tduc 215 122 32 Geospiza (dříve Gene Sifter) názorná demonstrace analýzy dat a interpretace výsledků (komerční software pro výzkumné účely vs. DAVID, Database for Annotation, Visualization and Integrated Discovery )
Importantly, the extremely high turnover and remodeling rate of matrix proteins (including collagens) is also associated with invasive cancer, which can be considered as a never healing wound (Dvorak N Engl J Med 1986, Weigelt et al. Nat Rev Cancer 2005).
Migration through collagen 1 with asporin MDA-MB-231 cell starved for 8 hours Transwells coated by collagen fibrillogenesis for 6 hours (in 30 ul, with or without 10 nm recombinant asporin; xcelligence system, Roche) Assembling the CIM plate with chemoatractant in bottom well (10% FBS, 170 ul) Seeding cells on the collagen (75 thousands in 100 ul)
Přehled Projekt HUGO 2001 (Human genome organization) První genomická vlna Expresní microarrays SNP (single nucleotide polymorphism), CGH arrays Druhá genomická vlna Genome-scale RNAi Next generation sequencing 2005/6 World class projects 1000 genomes ENCODE 2012 (Encyclopedia of DNA Elements)
Gene Expression Omnibus GEO Datasets search diagnosis/cell type GDS, curated gene expression dataset GSE, original submitted series (series of GSM, samples) (GPL, platform) GEO Profiles (from GDS) search gene of interest
integrated array data from more than 5,300 human samples from 163 different labs, representing more than 350 cell and tissue types, disease states, and cell lines. The researchers identified six distinct major groups, or "continents," of gene expression: brain; muscle; blood-related; healthy and tumor solid tissues; cell lines derived from solid tissues, and partially differentiated cells.
Selected databases GEO, Gene Expression Omnibus (Pubmed) mrna expression profiles, SNP arrays, microrna profiles, methylation profiles and others Interactive analysis Geo2R ArrayExpress (EBI, Cambridge) A global map of human gene expression Oncomine Broad CCLE (Cancer Cell Line Encyclopedia) www.ebi.ac.uk/arrayexpress www.ebi.ac.uk/gxa/array/u133a www.oncomine.org www.broadinstitute.org/ccle/home
Přehled Projekt HUGO 2001 (Human genome organization) První genomická vlna Expresní microarrays SNP (single nucleotide polymorphism), CGH arrays Druhá genomická vlna Genome-scale RNAi Next generation sequencing 2005/6 World class projects 1000 genomes ENCODE 2012 (Encyclopedia of DNA Elements)
RNAi screens and other high-throughput technologies: online resources Jan Bouchal, Ph.D. Department of Clinical and Molecular Pathology www.researcherid.com/rid/a-3859-2008 Datalab open laboratory of biotechnological applications
RNA interference The ways in which mirnas cause silencing of their target mrnas are still debated. The mechanisms involved are likely to include: inhibition of translation; triggering removal of the poly(a) tail from mrnas (deadenylation); disruption of cap tail interactions; and degradation of mrnas by exonucleases. http://www.nature.com/nrg/multimedia/rnai/index.html
Small interfering RNAs Nature Reviews Genetics / Arkitek http://www.nature.com/nrg/multimedia/rnai/index.html
Dicer double-stranded-rna-specific ribonuclease Nature Reviews Genetics / Arkitek http://www.nature.com/nrg/multimedia/rnai/index.html
RISC RNA-induced silencing complex Nature Reviews Genetics / Arkitek http://www.nature.com/nrg/multimedia/rnai/index.html
Genome-scale RNAi screening This technology has been hailed as the second genomics wave, following the first genomics wave of gene expression microarray and single nucleotide polymorphism discovery platforms. Mohr et al. 2010 Annu. Rev. Biochem. 79:37 64
Endoribonuclease prepared (e)sirnas
False discoveries and validation RNAi atlas (http://rnaiatlas.ethz.ch)
Interactive on-line databases Mitocheck database (http://www.mitocheck.org) GenomeRNAi (http://www.genomernai.de/genomernai/) manual curation of RNAi screening data from literature (310 screens, 14 libraries, 500 000 phenotypes)
ENCODE (Encyclopedia of DNA Elements) ENCODE (link to Genome browser and other resources) http://encodeproject.org/encode/ http://www.nature.com/encode/ Genome browser free tutorial http://openhelix.com/cgi/tutorialinfo.cgi?id=27 Experiment matrix (plus link ChIP-seq Experiment Matrix) http://encodeproject.org/encode/datamatrix/encodedatamatrixhuman. html Complete epigenomes http://epigenomegateway.wustl.edu/browser/roadmap/ http://www.genome.gov/27551933