Ectopic expression of a MYB transcription factor defines a set of co-regulated genes involved in phenylalanine and phenylpropanoid synthesis in tomato fruit

Hybridization: PhODO1-8117/M82 (Breaker)

Hybridization protocol
Hybridization solutionNCSU Hyb Soln
Solution concentration30% formamide, 5X SSC, 5X Denhardts, 1%PolyA RNA (10ug/uL)and 0.1%SDS
Blocking agentN/A
Wash procedureSlides are washed twice in 1X SSC with 0.2% SDS(pre-heated to 43C) for 3 minutes. Next they are washed for 3 minutes in room temperature 0.1X SSC with 0.2% SDS. Slides were then dipped in room temperature 0.1X SSC with no SDS before washing 3 times with room temperature 0.1X SSC with no SDS for 3 minutes.
Labeled Target Quantity200pmol of each sample
Hybridization TimeOvernight (16hr)
Hybridization Volume65uL
Hybridization Temperature42C
Hybridization InstrumentCorning microarray hybridization chamber in 42C 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