trancriptome profiling of pennellii ILs
Hybridization: 5-5/M82
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Hybridization protocol
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| Hybridization solution | Genisphere Hybridization Solution |
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| Solution concentration | 0.25m NaPO4, 4.5%SDS, 1mm EDTA, 1x SSC, 2x Denhardt's Solution |
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| Blocking agent | ssDNA and Oligo dT |
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| Wash procedure | 3x15 minutes:
1) 2x SSC + 0.2% SDS at 60 C.
2) 2x SSC at 22 C.
3) 0.2x SSC at 22 C |
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| Labeled Target Quantity | 3 ul cDNA |
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| Hybridization Time | 12-16 hours |
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| Hybridization Volume | 60 ul |
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| Hybridization Temperature | 60C |
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| Hybridization Instrument | 50 ml tube in 60C chamber |
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Scan information
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| Scan hardware | Scan Array 5000 |
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| Scan software | Scan Array ver 3.1 |
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Image analysis information
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| Software | ImaGene ver 5.5.4 |
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| Analysis algorithm | 1) Images
Cy5 = Red Cy3 = Green
2) Grid Flexibility
Enforce grid constraints = yes
Local flexibility = 6.0 pixels
Flexibility = 75%
3) Quality Control
Empty spots = no
Poor spots = yes
Find negative spots = no
4) Measurements
Background buffer = 2.25
Background width = 5.0
Auto seqmentation = no
Signal low cutoff = 20%
Signal high cutoff = 100%
Background low cutoff = 0%
Background high cutoff = 88%
5) Poor Spot Parameters
Contamination of Background/Signal = no
Ignored pixels % = yes (setting = 69)
Open perimeter = no
Shape regularity = no
Area to perimeter = yes (setting = 0.6)
Off-set value = yes (setting = 17.5) |
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Normalization information
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| Normalization software | marray package under R |
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| Normalization algorithm | Print-tip specific Lowess Normalization |
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| Normalization strategy | Total Array |
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