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This workshop by Alex Pozhitkov, Peter A. Noble, and Diethard Tautz in 2011 delves into the conundrum of signal intensity against variables such as GC content and sense/antisense strands. Their study confirms the unpredictability of rRNA hybridization tests and explores factors like overhangs, buffer composition, and melting temperatures in microarray analysis instruments. The research emphasizes the importance of calibration in detecting copy number variations and acknowledges the support from the Max Planck Society and Alabama State University.
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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. 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
Melting study • Overhangs • Buffer composition • Tm Sense vs. Tm antisense • Tm vs. Signal Intensity on the array
Tm Antisense (0C) Tm Sense (0C) Melting Temperatures
Microarray is an Instrument Instruments require calibration
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
Freundlich isotherm!y=axb Langmuir isotherm is not the only possible isotherm: Pozhitkov et al. (2010) Beyond Affymetrix arrays: … NAR 38, e28.
DNA and RNA y=axb+c
Measurements and Errors Single probe replicate Average over 10 replicates
CNV detection CNV expected: 1
Sample prep variability $ loci
Acknowledgements • Max Planck Society • Alabama State University