Upload
avis-sullivan
View
227
Download
1
Tags:
Embed Size (px)
Citation preview
Gene Expression Gene Expression DataData
Qifang XuQifang Xu
OutlineOutline
cDNA Microarray TechnologycDNA Microarray Technology Data RepresentationData Representation Statistical Analysis of cDNA Statistical Analysis of cDNA
Microarray DataMicroarray Data Oligonucleotide Microarray Oligonucleotide Microarray
TechnologyTechnology Microarray ApplicationsMicroarray Applications Microarray DatabasesMicroarray Databases
cDNA MicroarraycDNA Microarray
Measure the relative levels of Measure the relative levels of expression expression
Parallel analysisParallel analysis Competitive hybridizationCompetitive hybridization Need cDNA libraryNeed cDNA library
mRNA cDNAmRNA cDNA
Reverse Transcription
PCR Amplification
Printing
Hybridization
Laser Scan
Labeling
SamplesReverse Transcription
Expression Data
Exponential Amplification of a Exponential Amplification of a GeneGene
Return
Labeling and Hybridization Labeling and Hybridization
of Sample cDNAsof Sample cDNAs
Return
Data DescriptionData Description Ratios, why?Ratios, why?
Competitive hybridization Competitive hybridization no absolute expression levels no absolute expression levels
Gene expression levels determined by intrinsic properties of each gene
low high expression level
Gene A Gene B
Sample 2
Sample 1
Data NormalizationData Normalization
PurposePurposeAdjust bias from variation in microarray Adjust bias from variation in microarray technology.technology.
E.g. differences between labeling, scanner setting, E.g. differences between labeling, scanner setting, spatial positionsspatial positions
Within-array normalizationWithin-array normalizationlogarithmic transformation of ratio, subtract by logarithmic transformation of ratio, subtract by mean log ratiomean log ratio
Red Green Difference Ratio (G/R) Log2 Ratio Centered R
16500 15104 -1396 0.915 -0.128 -0.048
357 158 -199 0.443 -1.175 -1.095
8250 8025 -225 0.973 -0.039 0.040
978 836 -142 0.855 -0.226 -0.146
65 89 24 1.369 0.453 0.533
684 1368 529 2.000 1.000 1.080
13772 11209 -2563 0.814 -0.297 -0.217
856 731 -125 0.854 -0.228 -0.148
Statistical AnalysisStatistical Analysis
• Differences in ratios due to– random variation
– meaningful changes
• Convention
– ratio >= 2 or ratio <= ½
• Analysis of variance (ANOVA)– 4 and 10 replicates of each treatment
– statistical significance
Oligonucleotide Oligonucleotide MicroarrayMicroarray
Parallel analysis of gene expressionParallel analysis of gene expression Unit of hybridization: oligonucleotidesUnit of hybridization: oligonucleotides
represent known or predicted open reading frames (ORF)represent known or predicted open reading frames (ORF)
Noncompetitive hybridizationNoncompetitive hybridization Data RepresentationData Representation
average differenceaverage difference between perfect match and mismatch between perfect match and mismatch intensitiesintensities
Difference in expression levels between Difference in expression levels between two samplestwo samplescomparison across chipscomparison across chips
Oligonucleotide Oligonucleotide MicroarrayMicroarray
Construction of Construction of oligonucleotide arraysoligonucleotide arrays
Comparison between cDNA and Comparison between cDNA and Oligonucleotide MicroarrayOligonucleotide Microarray
cDNA MicroarraycDNA Microarray cheaper cheaper replicates replicates hybridization over kilobases (1 – 2 kb)hybridization over kilobases (1 – 2 kb)
reduce cross-hybridizationreduce cross-hybridization genome sequence unknowngenome sequence unknown cDNA librarycDNA library Competitive hybridizationCompetitive hybridization
Oligonucleotide MicroarrayOligonucleotide Microarray accommodate higher densities of genes accommodate higher densities of genes
predicted genes not in cDNA predicted genes not in cDNA librarylibrary
lower variabilitylower variability incorporate mismatch controls incorporate mismatch controls
reduce possibility of cross-reduce possibility of cross-hybridizationhybridization
Sequence knownSequence known Noncompetitive hybridizationNoncompetitive hybridization
Microarray ApplicationsMicroarray Applications
Drug Discovery – Identification of drug targets to treat diseases
Drug Development – rule out potential drug candidates before entering trials
Diagnostics and Disease Treatment – Screening and identification of patient samples
Model Organismsmouse brain modelLife cycle of a fruitfly
Genetic Regulatory NetworkGenetic Regulatory Network
Gene Expression Gene Expression DatabasesDatabases
Stanford Microarray Database (SMD)Stanford Microarray Database (SMD) http://genome-www5.stanford.edu/MicroArray/SMhttp://genome-www5.stanford.edu/MicroArray/SM
D/D/ raw and normalized data, image filesraw and normalized data, image files
The Gene Expression Database (GXD)The Gene Expression Database (GXD) http://http://
www.informatics.jax.org/mgihome/GXD/aboutGXD.www.informatics.jax.org/mgihome/GXD/aboutGXD.shtmlshtml
Endogenous gene expression during mouse Endogenous gene expression during mouse development.development.
ExpressDB ExpressDB http://http://twod.med.harvard.edu/ExpressDBtwod.med.harvard.edu/ExpressDB// Relational database for yeast and E. coli RNA Relational database for yeast and E. coli RNA
expression dataexpression data
Thank you !Thank you !