Time course of DC3000 vs delta hrp on tomato Prf-3

Hybridization: DC3000/delta hrp 6h

Hybridization protocol
Hybridization solutionNEN buffer Q
Solution concentrationNEN hybridization buffer Q
Blocking agentuknown-in commercial buffer
Wash procedure3X 15 minutes each:
1) 0.5X SSC, 0.01% SDS, at RT.
2) 0.06X SSC, 0.01% SDS at RT.
3) 0.06X SSC at RT
Labeled Target Quantity50ug RNA
Hybridization Time12-16 hours
Hybridization Volume65 ul
Hybridization Temperature60 C
Hybridization InstrumentCorning microarray hybridization chamber

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