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Direct calibration of microarray probes

Direct calibration of microarray probes. Making a “DNA-meter”. Alex Pozhitkov, Peter A. Noble, Diethard Tautz, Ploen Workshop, 2011. Conundrum. Signal intensity vs. GC Signal intensity vs. D G 0 Sense vs. antisense (same genomic loci) R 2 =0.37. Confirms:

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Direct calibration of microarray probes

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  1. Direct calibration of microarray probes Making a “DNA-meter” Alex Pozhitkov, Peter A. Noble, Diethard Tautz, Ploen Workshop, 2011.

  2. Conundrum • Signal intensity vs. GC • Signal intensity vs. DG0 • Sense vs. antisense (same genomic loci) R2=0.37 Confirms: Pozhitkov, et al (2006). Tests of rRNA hybridization … cannot be predicted. NAR 34, e66-e6

  3. Melting study • Overhangs • Buffer composition • Tm Sense vs. Tm antisense • Tm vs. Signal Intensity on the array

  4. Tm Antisense (0C) Tm Sense (0C) Melting Temperatures

  5. Microarray is an Instrument Instruments require calibration

  6. 800 5000 5000 4000 600 3000 400 2000 200 1000 8 6 2 0 4 8 6 2 0 4 “Good”, “Bad” and Noise Signal Intensity Concentration Individual replicas Averages

  7. Freundlich isotherm!y=axb Langmuir isotherm is not the only possible isotherm: Pozhitkov et al. (2010) Beyond Affymetrix arrays: … NAR 38, e28.

  8. DNA and RNA y=axb+c

  9. Measurements and Errors Single probe replicate Average over 10 replicates

  10. CNV detection CNV expected: 1

  11. Sample prep variability $  loci

  12. Acknowledgements • Max Planck Society • Alabama State University

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