How DNA Microarrays Work

1. Exploring the Limitations of DNA Microarray Technology: A Case Study in Quality Control Emily Oldham1, 2,A. Malcolm Campbell1, Laurie J. Heyer 2 1Biology & 2Math Depts., Davidson College, Davidson, NC 7 4 1 How DNA Microarrays Work Microarray Production Ratios from Alternative Methods Step 1: Spot each gene onto microarray Step 2: cDNAs from two conditions are labeled red and green Step 3: Hybridize cDNAs to microarray Step 4: Scan to measure fluorescence Step 5: Determine red:green ratios A schematic map of the experimental microarray spots. Each colored box represents a different known yeast (or fly) PCR product. Diagonals show a 4096-fold concentration range. Theoretical versus actual experimental ratios for three hybridization methods. No method gave 1:1 ratios consistently. Error bars are 90% confidence intervals. 2 5 8 Data Collection Data Analysis Why Microarrays are Exciting • Genome-wide expression of genes can be determined quickly • Current applications include: • - cellular circuitry • - drug discovery • - mutation and polymorphism detection Differences between pairwise ratios are gene- and method-dependent. Error bars are 90% confidence intervals. A microarray hybridized for all 10 yeast genes in equal concentrations of both red and green dye. http://www.tigr.org/tdb/microarray/images.shtml 6 3 9 Research Goals Gene-Specific Ratios Conclusions • Some genes will give unpredictable ratios • Rigorous controls should: • a) identify any aberrant genes • b) compensate for gene-specific ratios • Significance for ratio values must be determined for each microarray application • Design novel system to evaluate microarray method • Determine if microarrays detect known 1:1 ratios • Measure gene-specific variation in ratios • Document sources of experimental error Theoretical versus actual experimental results. Ratios were filtered for spot morphology and 15% similarity between partner spots. Values for the three highest concentrations and three experimental replicates were pooled. emoldham@davidson.edu Helping Students Discover Genomics, Proteomics & Bioinformatics A. Malcolm Campbell, Laurie J. Heyer, Adam Abele, Brian Akin, Danielle Choi, Parul Karnik, Peter Lowry, David Moskowitz, Emily Oldham, Jennifer Madden