Statistical Issues in the Design of Microarray Experiment Jean Yee Hwa Yang University of California San Francisco http www biostat ucsf edu jean Stat 246 Lecture 18 Mar 30 2004 SAGE Nylon membrane Illumina Bead Array Different Technologies GeneChip Affymetrix cDNA microarray Agilent Long oligo Ink Jet CGH From W Gibbs Scientific American 2001 Life Cycle Biological question Experimental design Failed Microarray experiment Quality Measurement Image analysis Preprocessing Normalization Pass Analysis Estimation Testing Clustering Biological verification and interpretation Discrimination Experimental design Proper experimental design is needed to ensure that questions of interest can be answered and that this can be done accurately given experimental constraints such as cost of reagents and availability of mRNA Definition of probe and target TARGET PROBE cDNA A Cy5 labeled cDNA B Cy3 labeled Two main aspects of array design Design of the array Allocation of mRNA samples to the slides cDNA A Cy5 labeled cDNA B Cy3 labeled Arrayed Library 96 or 384 well plates of bacterial glycerol stocks Spot as microarray on glass slides Hybridization Two main aspects of array design Design of the array Allocation of mRNA samples to the slides WT MT Hybridization Some aspects of design 1 Layout of the array Which 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 These relates to preprocessing such as quality assessment and normalization External Controls External controls From Van de Peppel et al 2003 From Van de Peppel et al 2003 References Microarray sample pool Yang et al 2002 Normalization for cDNA microarray data a robust composite method addressing single and multiple slide systematic variation Nucleic Acids Research Vol 30 No 4 e15 Titrated external controls J van de Peppel et al 2003 Monitoring global mRNA changes with externally controlled microarray experiments Embo Reports 4 387 393 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 Some aspects of design 2 Allocation of samples to the slides A Types of Samples Replication technical biological Pooled vs individual samples Pooled vs amplification samples This relates to both Affymetrix and two color spotted array 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 Avoidance of bias Conditions of an experiment mRNA extraction and processing the reagents the operators the scanners and so on can leave a global signature in the resulting expression data Randomization Local control is the general term used for arranging experimental material Preparing mRNA samples Mouse model Dissection of tissue RNA Isolation Amplification Probe labelling Hybridization Preparing mRNA samples Mouse model Dissection of tissue RNA Isolation Amplification Probe labelling Hybridization Biological Replicates Preparing mRNA samples Mouse model Dissection of tissue RNA Isolation Amplification Probe labelling Hybridization Technical replicates Preparing mRNA samples Mouse model Dissection of tissue RNA Isolation Amplification Probe labelling Hybridization Technical replicates Technical replication amplification Olfactory bulb experiment 3 sets of Anterior vs Dorsal performed on different days 10 and 12 were from the same RNA isolation and amplification 12 and 18 were from different dissections and amplifications All 3 data sets were labeled separately before hybridization Data provided by Dave Lin Cornell Pooling looking at very small amount of tissues Mouse model Dissection of tissue RNA Isolation Pooling Probe labelling Hybridization Design 1 Pooled vs Individual samples Design 2 Taken 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 hybridization Pooling can assists reducing the number of hybridization Pooled vs amplified samples amplification amplification Design A amplification amplification pooling Design B pooling Original samples Amplified samples Pooled vs Amplified samples In the cases where we do not have enough material from one biological sample to perform one array chip hybridizations Pooling or Amplification are necessary Amplification Introduces more noise Non linear amplification different genes amplified at different 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 study of the effects of pooling on gene expression estimates using high density oligonucleotide array data Department of Biological Science University of Tulsa February 2003 Kendziorski C M Y Zhang H Lan and A D Attie 2003 The efficiency of mRNA pooling in microarray experiments Biostatistics 4 465 477 7 2003 Xuejun Peng Constance L Wood Eric M Blalock Kuey Chu Chen Philip W Landfield Arnold J Stromberg 2003 Statistical implications of pooling RNA samples for microarray experiments BMC Bioinformatics 4 26 6 2003 Some aspects of design 2 Allocation of samples to the slides 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 limitation or cost into consideration the number of slides the amount of material Graphical representation Vertices mRNA samples Edges hybridization Direction dye assignment Cy3 sample Cy5 sample Graphical representation The structure of the graph determines which effects can be estimated and the precision of the estimates Two mRNA samples can be compared only if there is a path joining the corresponding two vertices The precision of the estimated contrast then
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