Tomato (breaker-stage) vs. Pepper (breaker-stage)

Hybridization: Tomato Breaker/Pepper Breaker

Hybridization protocol
Hybridization solutionCorning Universal Hybridization Solution (catalog #40090, Corning Inc.)
Solution concentration Unknown due to Corning patent
Blocking agentUnknown due to Corning patent
Wash procedure1) Wash arrays in 1X SSC/0.2% SDS for 10 minutes at 43 C (in the dark).
2) Wash arrays in 0.1X SSC/0.2% SDS for 10 minutes at room temperature (in the dark, with shaking).
3) Wash arrays in 0.1X SSC for 10 minutes at room temperature (in the dark, with shaking).
Labeled Target QuantityAll (see instructions in Invitrogen labeling kit)
Hybridization Time12 - 16 hours
Hybridization Volume65 ul
Hybridization Temperature43C
Hybridization InstrumentCorning microarray hybridization chamber in 43C incubator

Scan information
Scan hardwareScan Array 5000
scan softwareScan Array ver 3.1

Image analysis information
SoftwareImaGene ver 5.5
Analysis algorithm1) Images
Cy5 = Red Cy3 = Green

2) Spot Finding
Find negative spots = no
Enforce grid constraints = yes
Local flexibility = 6.0
Global flexibility = 75%

3) Segmentation
Background buffer = 2.25
Background width = 5.0
Autosegmentation = no
Signal low cutoff = 20%
Signal high cutoff = 100%
Background low cutoff = 0%
Backgound high cutoff = 88%

4) Quality Flags
Empty spots = no
Negative spots = yes
Poor spots = yes

5) Poor Spot Parameters
Contamination of background/signal = no
Ignored pixels percentage = yes (setting = 69%)
Open perimeter ratio = no
Shape regularity = no
Area to perimeter ratio = yes (setting = 0.6)
Offset value = yes (setting = 17.5)

Normalization information
Normalization softwaremarray package under R
Normalization algorithmPrint-tip specific Lowess Normalization
Normalization strategyTotal Array