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Experiment Design for Affymetrix Microarray. Affymetrix Terminology. Probe: A 25mer oligo complemetary to a sequence of interest, attached to a glace surface on the probe array. Perfect Match: (PM) Probes that are complementary to the sequence of interest.
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Experiment Design for Affymetrix Microarray
Affymetrix Terminology Probe:A 25mer oligo complemetary to a sequence of interest, attached to a glace surface on the probe array Perfect Match: (PM) Probes that are complementary to the sequence of interest. Mismatch : (MM)Probes that are complementary to the sequence of interest except for homomeric base change (A-T or G-C) at the 13th position Probe Pair: (PP) A combination of a PM and MM; 11-16 probe pairs/ probe set Probe Cell: A single feature; size can be 18X18 or 20X20u
Experimental Design Flow Simplified Data Analysis Pilot Study Full Scale Experiment Publication Bioinformatics Data Validation Complete Analysis
Advantages of a Pilot Study • Estimate experimental variability • Refine laboratory methods/techniques • Refine experimental design • Allows for rapid screening • Provides preliminary data for project funding
Three Sources of Variability • Biological : Differences between samples • - The ultimate goal of the research • Technical: Sample preparation • - Protocols and operator • System: Probe Array analysis • - Arrays, instruments, reagents
Controlling Biological Variability • Biological variability contributes more to experimental variability • than technical variability. • To mitigate biological variability:- • - Consider all potential variables as part of the experiment design • - Increase the number of biological replicates until Coefficient of • Variation (CV) stabilizes
Examples of Biological Variability • Cell Cycle Patterns- What time of day were the samples isolated? • Circadian Rhythm- What is the time interval between time course samples? • Nutrient- Media types will affect expression levels • Tissue- Each cell type has different expression pattern • Temperature- Growth room temperature may vary within a 24h period • Disease- Defense genes will alter global gene expression pattern • Germination time- Different seed batches will alter gene expression pattern
Practical Questions to Consider • How much variability does your system have? • - Understand and minimize variation • What level of significance is needed? • - More replicates needed for subtle changes • How many treatments? How many controls? • - Comparative analysis (one experimental condition) or serial analysis • design (multiple experimental conditions)?
Percentage CV as Estimate of Variability • CV% is a measure of variance amongst replicates of a single condition • Defined as the standard deviation divided by the mean multiplied by 100 • Example: 6 signal values representing 6 replicates • - 230.4, 241.7, 252.9, 338.8, 178.9, 339.6 • - Mean = 263.72; = 63.72; CV% = 24.16%
Experimental Replicates • Technical replicates from the same sample reproduce the contribution • from the bench effects to the overall variability • Biological replicates: “True” replicates that reproduce biological conditions • explored in the experimental design • - Permit the use of formal statistical tests • - Also allows the interrogation of technical variability
RNA Sample Pooling • Can increase sample quantity • A common variance mitigation strategy • Can result in irreversible loss of information by introducing a bias • If necessary pool a minimum of three or a maximum of five RNAs • Equal pooling of RNA samples is essential
Why Normalize ? • To correct for systematic measurement error and bias in data • Allows for data comparison