Results Davidson College Biology Department DNA Microarrays: A guide to teaching chips Allison Amore, Sheena Bossie, Max Citrin, Erin Cobain, Megan McDonald, Marieta Solé, Emily Wilson A. Malcolm Campbell 5 DNA microarrays measure expression level changes across a genome and have the potential to be a powerful diagnostic tool in medicine. Equipment necessary to print DNA microarrays is expensive & complex Undergraduates have little experience with this technique Undergraduates face challenge of learning about DNA microarray technology without hands on experience PROBLEM TO BE ADDRESSED: How can DNA microarray technology be introduced at the undergraduate level in a laboratory setting? POTENTIAL SOLUTION  Teaching Chips - provide students with thorough understanding of microarray methodology but do not necessitate the use of expensive RNA samples or genomic DNA. Students learn how to design, print (if the equipment is available) and process chips that are likely to have a high success rate with visible data. Setup Idh1 We designed a stoplight pattern using three S. Cerevisiae genes (Mhp, Idh1, and SHY). Competent bacterial cells containing plasmids with 500bp fragments of genes were obtained from a previous study (D. Pierce). Genes were chosen because their probes had been previously shown not to bind to either of the other two genes (D. Pierce). Design was printed using BioRobotics MicroGrid II Compact  We followed the 2-step 3DNA Genisphere  for probe hybridization (previously optimized by E. Oldham) The Microarray Process Essential Steps: grow cells containing RNA of interest isolate and extract RNA convert mRNA into cDNA spot cDNA onto slide hybridize with Cy3 and Cy5 tagged probes scan chip to measure fluorescence intensity analyze data to determine repression and induction patterns from image profiles. 3DNA dendrimer courtesy of Genisphere, Inc. Gene Systematic NameChromosomeBrief Description of Function MHP1YJL042WXmicrotubule organization IDH1YNL037CXIV Regulatory subunit in cellular energy metabolism SHY1YGR112WVII Codes for a mitochondrial protein required for full expression of cytochrome oxidase (COX) QIAGEN HiSpeed Plasmid Midi Kit Promega PureYield Plasmid Midiprep kit List price $250 for 25 prepsList price $180 for 25 preps Requires little centrifugation, FasterMultiple centrifugation steps, Slower Slightly more consistent resultsSlightly greater variability Introduction Future Work Perform experiment using oligos with correct capture sequence. Confirm results using Q-PCR Analyze data using Magic Tool Compose a single detailed protocol Promote the use of teaching chips in undergraduate settings Publish work in Cell Biology Education Fluorescent labeling process using Cy5 (red) and Cy3 (green) dyes: (Cartoon created by E. Oldham) Part I: Two different plasmid isolation kits were compared: Results Continued Part III: Prepared test slide to determine whether all reagents were functioning properly: Test Slide Design. Anticipated results if all reagents were functioning properly. O = oligo, RT = Reverse Transcript, C = control. Actual Test Slide Results. Only the two positive controls fluoresced as expected. CONCLUSION: All proposed reasons for our negative results were proven false. The unexpected outcome was due to a capture sequence that was not complementary to the dendrimer sequence! Part II: Chips were scanned to visualize stoplight pattern  no fluorescence was detected on any of the slides. Potential Reasons for this Outcome: Fluorescent dyes have degraded and lost ability to fluoresce Oligos were non-functional or degraded Plasmid DNA may not have been isolated properly SHY idh1 Mhp SHY Mhp 120 ng/ul 240 ng/ul SHY-O SHY-O Mhp-O Mhp-O idh1-O idh1-O RT-C RT-C Acknowledgements We would like to thank the following people for their invaluable support: Dr. A. Malcolm CampbellDanielle Choi Emily OldhamDavidson College Biology Dept. Dan PierceVarious service technicians Chris Healey