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. G0
• 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
Melting Temperatures
Tm Sense (0C)
Tm
Ant
isen
se (
0 C)
Microarray is an InstrumentInstruments require calibration
“Good”, “Bad” and Noise
Individual replicas Averages
800
600
400
200
0 2 4 6 8
50005000
4000
3000
2000
1000
0 2 4 6 8
Concentration
Sig
nal
In
ten
sity
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