microarray normalization

1
microarray normalization

• Slides courtesy of Wolfgang Huber, Jean Yang ,
  Terry Speed, Eric Rouchka, Gary Churchill

2
excitation
scanning
cDNA clones (probes)
laser 1
laser 2
PCR product amplification purification
emission
printing
mRNA target)
overlay images and normalise
0.1nl/spot
Hybridise target to microarray
microarray
analysis
3
Steps in Images Processing
1. Addressing locate centers
2. Segmentation classification of pixels either
as signal or background.
3. Information extraction for each spot of the
array, calculates signal intensity pairs,
background and quality measures.
4
Steps in Image Processing
3. Information Extraction

• Spot Intensities
• median (pixel intensities).
• Background values
• median (pixel intensities).
• Reading
• Spot int. Bckg int.

Signal
Background
5
Data transformation is essential
6
Data transformation is essential
7
Log transform not always appropriate - vsn
normalization Huber et al.
8
residual plot - M vs. A plot
9
LOWESS Adjustment of M Values
M
A
10
Final result - M values after LOWESS Adjustment
M
A