Frozen digestive gland (DG) tissues, from mussels treated with heavy metals (cadmium, cupper, mercury) and control mussels, were dispersed separately into TRizol solution (Invitrogen, Carlsbad, CA, USA), and homogenized with an Diax 900 blender (Heidolph, Kelheim, Germany). Total RNA (15 µg), extracted following the manufacturer instructions, was reverse transcribed with SuperScript II (Invitrogen) and oligo-dT(18) as the primer, in the presence of either Cy3-dCTP or Cy5-dCTP (Amersham Biosciences, Uppsala, Sweden). Competitive hybridizations were carried out in a dual slide chamber (HybChamber, GeneMachines, San Carlos, CA, USA). Pellets of purified, labeled cDNAs were dissolved in hybridization buffer (Northern max, Ambion), applied on the MytArray 1.0, and covered with a 22×22 mm glass coverslip. After an overnight hybridization at 42°C, slides were washed sequentially for 4 min in buffer A (1X SSC/0.2% SDS) at 42°C, for 4 min buffer B (0.1X SSC/0.2% SDS) at room temperature and finally in 0.1X SSC for 2 min at room temperature. Digital images were generated in a GSI Lumonics LITE dual confocal laser scanner (ScanArray Microarray Analysis Software) and processed with QuantArray Analysis Software (GSI Lumonics, Ottawa, Canada). Normalized data were determined using the publicly available software MIDAS (TIGR Microarray Data Analysis System: http://www.tigr.org/softlab/). Normalized values (lowess normalization) were calculated for each spot, and converted in logarithmic scale. Final values correspond to log(2) ratio of the normalized intensities. Keywords = digestive gland Keywords = heavy metals Keywords = transcriptional profiling
