文档介绍：Image Processing for cDNA Microarray Data
Prepared with massive assistance from Yee Hwa Yang (Berkeley, WEHI), and reporting on work done jointly with her, Sandrine Dudoit (Stanford) and Mike Buckley (CSIRO, Sydney).
References : M Eisen and P Brown, Methods in Enzymology vol 303, 1999; Chapter 2, DNA Microarrays (ed M Schena, OUP 1999) by Mack J Schermer; Chapter 13, Microarray Biochip Technology (ed M Schena, Eaton 2000) by Basarsky et al.
Scanner Process
Dye
Photons
Electrons
Signal
Laser
PMT
A/D
Convertor
excitation
amplification
Filtering
Time-space
averaging
GenePix 4000a Microarray Scanner Protocol
1. Turn on scanner.
2. Slide scanner door open. Insert chip hyp side down and clip chip holder easily around the slide
3 Set PMTs to 600 in both 635nm (Cy3) and 532 (Cy5) channels.
4. Perform low resolution “PREVIEW SCAN” to determine location of spots and initial hyb intensities
5. Once scan location determined, draw a “SCAN AREA” marquis around the array
6. Perform quick visual inspection of hyb and make initial adjustments to PMTs
7. For gene expression hybs, raise or lower the red and green PMTs to achieve color balance
8. Before you perform your data scan, change “LINES TO AVERAGE” to 2.
9. Perform a high-resolution “DATA-SCAN”……(ctd)
10. Observe the histograms and make adjustments to PMTs.
11. Once the PMT level has been set so that the Intensity Ratio is near perform a “DATA SCAN” over “SCAN AREA” and save the results.
12. To save your image, select “SAVE IMAGES”.
13. Save as type=Multi-image TIFF files.
14. Once scanned and saved, you are ready to assign spot identities and calculate results.
Note: For us, normalization is performed later during data analysis, see next lecture.
GenePix 4000a Microarray Scanner Protocol, ctd
Scanner
Laser
PMT
Dye
Glass Slide
Objective Lens
Detector lens
Pinhole
Beam-splitter
How to adjust for PMT?
Cy3 Cy5
1 600 600
2 650 600
3 650 650
4 700 650
5 650 700
6 700 700
7 750 750
saturated
Ve