Statistical Issues in the Design of MicroarrayExperimentJean Yee Hwa YangUniversity of California, San Franciscohttp://www.biostat.ucsf.edu/jean/Stat 246 Lecture 18, Mar 30, 2004*****GeneChip AffymetrixcDNA microarrayNylon membraneAgilent: Long oligo Ink JetIllumina Bead ArrayCGHSAGEDifferentTechnologiesFrom W. Gibbs, Scientific American, 2001Biological verification and interpretationMicroarray experimentExperimental designImage analysisNormalizationBiological questionTestingEstimationDiscriminationAnalysisClusteringLifeCycleQuality MeasurementFailedPassPreprocessingExperimental designProper experimental design is needed toensure that questions of interest can beanswered and that this can be doneaccurately, given experimental constraints,such as cost of reagents and availability ofmRNA.cDNA “A”Cy5 labeledcDNA “B”Cy3 labeledPROBETARGETDefinition of probe and targetTwo main aspects of array designDesign of the arrayAllocation of mRNAsamples to the slidesArrayed Library(96 or 384-well plates of bacterial glycerol stocks)Spot as microarrayon glass slidescDNA “A”Cy5 labeledcDNA “B”Cy3 labeledHybridizationTwo main aspects of array designDesign of the array Allocation of mRNAsamples to the slidesHybridizationMTWTSome aspects of design1. Layout of the arrayWhich sequence to print?- Affymetrix – selecting the PM and MM’s.- cDNA Library – Riken, NIA etc- Selecting oligo probes:- Operon (commercial).- Aglient (commercial).- Illumina (commercial).- OligoPicker (Wang & Seed, Harvard)- ArrayOligoSelector (Bozdech etal, DeRisi Lab, UCSF)- OligoArray 1.0 (Rouillard, Herbert and Zuker)Controls:Normalization and quality checks.Number:Duplicate or replicate spots within a slide position.Commonly asked questions• Should we put duplicates on a slides.[Discussion in Smyth et al (2003)]• What should be the percentage of control spots?• Where should the control spots be placed? [Theserelates to preprocessing such as quality assessment andnormalization].ExternalControlsFrom Van de Peppel et al, 2003External controlsFrom Van de Peppel et al, 2003References• Microarray sample pool.- Yang et al (2002). Normalization for cDNA microarray data: arobust composite method addressing single and multiple slidesystematic variation. Nucleic Acids Research, Vol. 30, No. 4, e15.• Titrated external controls.- J. van de Peppel, et al (2003).!Monitoring global mRNA changeswith externally controlled microarray experiments. Embo Reports4: 387-393.• An example of doping controls for microarray.- http://genome-www.stanford.edu/turnover/supplement.shtml• Commercial- Lucidea Universal ScoreCard by Amersham.http://www1.amershambiosciences.com/A Types of Samples- Replication – technical, biological.- Pooled vs individual samples.- Pooled vs amplification samples.B Different design layout- Scientific aim of the experiment.- Robustness.- Extensibility.- Efficiency.Taking physical limitations or cost into consideration:- the number of slides.- the amount of material.Some aspects of design2. Allocation of samples to the slidesThis relates to bothAffymetrix and two color spotted array.Avoidance of bias• Conditions of an experiment; mRNA extractionand processing, the reagents, the operators, thescanners and so on can leave a “globalsignature” in the resulting expression data.• Randomization.• Local control is the general term used forarranging experimental material.Preparing mRNA samples:Mouse modelDissection oftissueRNAIsolationAmplificationProbelabellingHybridizationPreparing mRNA samples:Mouse modelDissection oftissueRNAIsolationAmplificationProbelabellingHybridizationBiological ReplicatesPreparing mRNA samples:Mouse modelDissection oftissueRNAIsolationAmplificationProbelabellingHybridizationTechnical replicatesPreparing mRNA samples:Mouse modelDissection oftissueRNAIsolationAmplificationProbelabellingHybridizationTechnical replicatesTechnical replication - amplificationTechnical replication - amplificationOlfactory bulb experiment:• 3 sets of Anterior vs Dorsal performed on different days• #10 and #12 were from the same RNA isolation andamplification• #12 and #18 were from different dissections and amplifications• All 3 data sets were labeled separately before hybridizationData provided byDave Lin (Cornell)Pooling: looking at very small amount of tissuesMouse modelDissection oftissueRNAIsolationPoolingProbelabellingHybridizationDesign 1Design 2Pooled vs Individual samplesTaken from Kendziorski etl al (2003)Pooled vs Individual samples• Pooling is seen as “biological averaging”.• Trade off between- Cost of performing a hybridization.- Cost of the mRNA samples.Cost or mRNA samples << Cost per hybridizationPooling can assists reducing the number ofhybridization.amplificationamplificationOriginal samples Amplified samplesamplificationamplificationpoolingpoolingDesign ADesign BPooled vs amplified samplesPooled vs Amplified samples• In the cases where we do not have enough material fromone biological sample to perform one array (chip)hybridizations. Pooling or Amplification are necessary.• Amplification- Introduces more noise.- Non-linear amplification (??), different genes amplified atdifferent rate.- Able to perform more hybridizations.• Pooling- Less replicates hybridizations.References• Pooling vs Non-Pooling- Han, E.-S., Wu, Y., Bolstad, B., and Speed, T. P. (2003). A studyof the effects of pooling on gene expression estimates using highdensity oligonucleotide array data. Department of BiologicalScience, University of Tulsa, February 2003.- Kendziorski, C.M., Y. Zhang, H. Lan, and A.D. Attie. (2003). Theefficiency of mRNA pooling in microarray experiments.Biostatistics 4, 465-477. 7/2003- Xuejun Peng, Constance L Wood, Eric M Blalock, Kuey ChuChen, Philip W Landfield, Arnold J Stromberg (2003). Statisticalimplications of pooling RNA samples for microarray experiments.BMC Bioinformatics 4:26. 6/2003A Types of Samples- Replication – technical, biological.- Pooled vs individual samples.- Pooled vs amplification samples.B Different design layout- Scientific aim of the experiment.- Robustness.- Extensibility.- Efficiency.Taking physical limitation or cost into consideration:- the number of slides.- the amount of material.Some aspects of design2. Allocation of samples to the slidesGraphical representationVertices: mRNA samples;Edges: hybridization;Direction: dye assignment. Cy3sampleCy5 sampleGraphical representation• The structure of the graph determines
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