Total RNA Extraction and cDNA Labeling

Total RNA was purified using phenol extraction [74] (link). Fluorescently labelled cDNA was prepared from total RNA using the SuperScript Plus Direct cDNA Labelling System (Life Technologies) as described by the manufacturer, except for the following modifications: 8 µg of total RNA was labelled in a reaction volume of 15 µl. 0.5 µl of 10× nucleotide mix with labelled nucleotide were used (1/3 of the recommended amount) and 1 µl of a home-made dNTP mix (0.5 mM dATP, 0.5 mM dCTP, 0.5 mM dGTP, 0.3 mM dTTP) was added to the reaction. All other components were used at the recommended concentrations. Note that these changes are essential to prevent dye-specific biases. Labelled cDNAs were hybridised to oligonucleotide microarrays manufactured by Agilent as described [63] (link). Microarrays were scanned with a GenePix 4000A microarray scanner and analysed with GenePix Pro 5.0 (Molecular Devices).