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