Maintenance and calibration of laser Doppler probes and pressure cuffs
Direct calibration of microarray probes
description
Transcript of Direct calibration of microarray probes
![Page 1: Direct calibration of microarray probes](https://reader036.fdocuments.us/reader036/viewer/2022062500/5681581b550346895dc57f47/html5/thumbnails/1.jpg)
Direct calibration of microarray probes
Making a “DNA-meter”
Alex Pozhitkov, Peter A. Noble, Diethard Tautz,Ploen Workshop, 2011.
![Page 2: Direct calibration of microarray probes](https://reader036.fdocuments.us/reader036/viewer/2022062500/5681581b550346895dc57f47/html5/thumbnails/2.jpg)
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
![Page 3: Direct calibration of microarray probes](https://reader036.fdocuments.us/reader036/viewer/2022062500/5681581b550346895dc57f47/html5/thumbnails/3.jpg)
Melting study
• Overhangs• Buffer composition• Tm Sense vs. Tm antisense• Tm vs. Signal Intensity on the array
![Page 4: Direct calibration of microarray probes](https://reader036.fdocuments.us/reader036/viewer/2022062500/5681581b550346895dc57f47/html5/thumbnails/4.jpg)
Melting Temperatures
Tm Sense (0C)
Tm
Ant
isen
se (
0 C)
![Page 5: Direct calibration of microarray probes](https://reader036.fdocuments.us/reader036/viewer/2022062500/5681581b550346895dc57f47/html5/thumbnails/5.jpg)
Microarray is an InstrumentInstruments require calibration
![Page 6: Direct calibration of microarray probes](https://reader036.fdocuments.us/reader036/viewer/2022062500/5681581b550346895dc57f47/html5/thumbnails/6.jpg)
“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
![Page 7: Direct calibration of microarray probes](https://reader036.fdocuments.us/reader036/viewer/2022062500/5681581b550346895dc57f47/html5/thumbnails/7.jpg)
Freundlich isotherm!y=axb
Langmuir isotherm is not the only possible isotherm:Pozhitkov et al. (2010) Beyond Affymetrix arrays: … NAR 38, e28.
![Page 8: Direct calibration of microarray probes](https://reader036.fdocuments.us/reader036/viewer/2022062500/5681581b550346895dc57f47/html5/thumbnails/8.jpg)
DNA and RNA
y=axb+c
![Page 9: Direct calibration of microarray probes](https://reader036.fdocuments.us/reader036/viewer/2022062500/5681581b550346895dc57f47/html5/thumbnails/9.jpg)
Measurements and Errors
Single probe replicate Average over 10 replicates
![Page 10: Direct calibration of microarray probes](https://reader036.fdocuments.us/reader036/viewer/2022062500/5681581b550346895dc57f47/html5/thumbnails/10.jpg)
CNV detection
CNV expected: 1
![Page 11: Direct calibration of microarray probes](https://reader036.fdocuments.us/reader036/viewer/2022062500/5681581b550346895dc57f47/html5/thumbnails/11.jpg)
Sample prep variability
loci
$
![Page 12: Direct calibration of microarray probes](https://reader036.fdocuments.us/reader036/viewer/2022062500/5681581b550346895dc57f47/html5/thumbnails/12.jpg)
Acknowledgements
• Max Planck Society
• Alabama State University