Petunia hybrida plants were grown in soil under normal greenhouse conditions.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated using Trizol reagent (Life Technologies, Cleveland, OH) according to the manufacturer instructions.
Label
Cy5
Label protocol
Total RNA (1μg) was used as a template to synthesize antisense RNA (aRNA) with the SuperScript™ Indirect RNA Amplification System Kit (Invitrogen) incorporating Alexa Fluor 647 Reactive Dye according to manufacturer's instructions
Hybridization protocol
Prehybridization was performed by incubating the arrays with prehybridization solution (6X SSPE, 0.05% Tween-20, 20mM EDTA, 5x Denhardt's solution, 100 ng/ul Salmon Sperm DNA, 0.05% SDS) for 60 minutes at 45 °C. 5 ug of labeled aRNA were fragmented by incubation with Fragmentation Solution (40mM Tris Acetate pH 8.1, 100 mM KOAc, 30 mM MgOAc) for 20 min at 95 °C. Hybridization was performed at 45°C for 16 hours in hybridization solution (6X SSPE, 0.05% Tween-20, 20mM EDTA, 25% DiFormamide, 100 ng/ul Salmon Sperm DNA, 0.04% SDS). Hybridization washings were performed as the following: - wash with 6X SSPET wash solution (6X SSPE, 0.05% Tween-20). - wash with 3X SSPET wash solution (3X SSPE, 0.05% Tween-20). - wash with 0.5X SSPET wash solution (0.5X SSPE, 0.05% Tween-20). - wash with PBST wash solution (2X PBS, 0.1% Tween-20). - 2 washes with 2X PBS
Scan protocol
After hybridization and washing, the microarray was dipped in imaging solution, covered with LifterSlip™, and then scanned using a Perkin Elmer ScanArray 4000XL and the accompanying acquisition software (ScanArray Express Microarray Analysis System v4.0). Multiple scanning at different PMT was provided for each hybridization.
Description
MYB5b 3
Data processing
Data extraction was carried out using CombiMatrix Microarray Imager software, and a quantile normalization of raw medians and log2 transformation were performed using SPSS v.15.0 software. Further statistical processing was performed using the SAM analysis (T-MEV 4.3)
