Mussels of average size (shell length 4.93 ± 0.17 cm) were placed in plastic tanks (5 mussels/tank, 1 litre ASW/mussel) and exposed for 48 hours to equimolar mixtures of Cd, Cu and Hg, prepared at 50, 100 and 200 nM concentration from chloride salts. We changed the water, food (Coralife® Sera, Heinsberg, Germany) and metal salts every 12 h and monitored regularly the mussel reactivity. Control mussels were maintained in a fourth tank in parallel.
Growth protocol
Adult mussels (Mytilus galloprovincialis, Lmk. 1819) of mixed sex were collected from a farming site offshore Alberoni (Venice area, North Italy), acclimatized in standard conditions (18±1 °C, 32‰ artificial sea water, ASW).
Extracted molecule
total RNA
Extraction protocol
Total RNA was purified from individual half-gill samples using the Trizol reagent (Invitrogen) in agreement to the manufacturer’s instructions and a further purification with LiCl was applied. Equal amounts of RNA obtained from the five control mussels were pooled and used as reference against individual RNAs from three treated mussels/tank in the competitive hybridisation to the DNA microarray slides.
Label
Cy5
Label protocol
Reference and test RNAs (10 µg) were separately incubated with a degenerated oligo-dT18 primer (10 min at 70°C) in a volume of 10 µl each, added to 20 µl of reaction mix (1x first-strand buffer, 400 U SuperScript II InvitrogenTM, 0.5 mM dATP, 0.5 mM dGTP, 0.5 mM dCTP, 0.3 mM TTP, 0.2 mM 5-(3-aminoallyl)-dUTP, 0.5 mM DTT) and reverse transcribed for 2 h at 42°C. RNA was removed from single-stranded cDNA with 3 µl of 1 N NaOH, 0.6 µl of 500 mM Na2-EDTA and the reaction mixture was then neutralized with 3 µl of 1 N HCl and 8.5 µl of 2 M HEPES. We used Microcon YM-30 (Amicon separation, MILLIPORE®) to remove buffer, unincorporated dNTPs and free amines. Finally, the cDNA samples were vacuum dried. Mono-functional NHS-esters of Cy3 or -Cy5 dyes (CyDye Post-Labeling Reactive Dye Pack, Amersham GE Healthcare) were covalently coupled to the aminoallyl-cDNA probes in DMSO for 1 h at room temperature in the dark. Then, the samples were quenched with 4.5 µl 4 M hydroxylamine for 15 min and purified with the GeneEluteTM PCR Clean-Up Kit (Sigma).
Mussels of average size (shell length 4.93 ± 0.17 cm) were placed in plastic tanks (5 mussels/tank, 1 litre ASW/mussel) and exposed for 48 hours to equimolar mixtures of Cd, Cu and Hg, prepared at 50, 100 and 200 nM concentration from chloride salts. We changed the water, food (Coralife® Sera, Heinsberg, Germany) and metal salts every 12 h and monitored regularly the mussel reactivity. Control mussels were maintained in a fourth tank in parallel.
Growth protocol
Adult mussels (Mytilus galloprovincialis, Lmk. 1819) of mixed sex were collected from a farming site offshore Alberoni (Venice area, North Italy), acclimatized in standard conditions (18±1 °C, 32‰ artificial sea water, ASW).
Extracted molecule
total RNA
Extraction protocol
Total RNA was purified from individual half-gill samples using the Trizol reagent (Invitrogen) in agreement to the manufacturer’s instructions and a further purification with LiCl was applied. Equal amounts of RNA obtained from the five control mussels were pooled and used as reference against individual RNAs from three treated mussels/tank in the competitive hybridisation to the DNA microarray slides.
Label
Cy3
Label protocol
Reference and test RNAs (10 µg) were separately incubated with a degenerated oligo-dT18 primer (10 min at 70°C) in a volume of 10 µl each, added to 20 µl of reaction mix (1x first-strand buffer, 400 U SuperScript II InvitrogenTM, 0.5 mM dATP, 0.5 mM dGTP, 0.5 mM dCTP, 0.3 mM TTP, 0.2 mM 5-(3-aminoallyl)-dUTP, 0.5 mM DTT) and reverse transcribed for 2 h at 42°C. RNA was removed from single-stranded cDNA with 3 µl of 1 N NaOH, 0.6 µl of 500 mM Na2-EDTA and the reaction mixture was then neutralized with 3 µl of 1 N HCl and 8.5 µl of 2 M HEPES. We used Microcon YM-30 (Amicon separation, MILLIPORE®) to remove buffer, unincorporated dNTPs and free amines. Finally, the cDNA samples were vacuum dried. Mono-functional NHS-esters of Cy3 or -Cy5 dyes (CyDye Post-Labeling Reactive Dye Pack, Amersham GE Healthcare) were covalently coupled to the aminoallyl-cDNA probes in DMSO for 1 h at room temperature in the dark. Then, the samples were quenched with 4.5 µl 4 M hydroxylamine for 15 min and purified with the GeneEluteTM PCR Clean-Up Kit (Sigma).
Hybridization protocol
Equal amounts of Cy3- or Cy5-reference and Cy5- or Cy3-test samples were combined in the same tube and ethanol-precipitated. After re-suspension in 18 µl of hybridisation buffer (5x SSC, 50% formamide, 0.1% SDS) and denaturation for 3 min at 70°C, the Cy3/Cy5-coupled samples were competitively hybridised to the DNA microarray slides, covered with a 22×22 mm cover-slip and incubated overnight at 42°C in a humidified dual-slide chambers (HybChamber, GeneMachines).
Scan protocol
Slides were examined with a GSI Lumonics LITE dual confocal laser scanner at 5 µm resolution. Image processing and quantification of the fluorescence signals were performed by the ScanArray Express® software (PerkinElmer), at 570 and 670 nm for Cy3 and Cy5 detection, respectively.
Data processing
Normalisation of the fluorescence signals was performed by using the total and LOWESS (logfit) algorithm with MIDAS (http://www.tigr.org/software).
