ecotype background: Col-0 genotype/variation: COPT1 over-expressor tissue: 7 days seedling growth condition: MS withCupper
Treatment protocol
Seeds of Arabidopsis thaliana, ecotype Columbia-0 (Col-0) and the transgenic lines COPT1OE were surface-sterilized and stratified for 2 d at 4°C and were germinated in ½ MS medium plates including 1% sucrose (Murashige, 1962) supplemented or not with the indicated concentrations of cupper. In order to know the effects of both Cu, the components of the ½ MS medium were prepared separately according to the conditions: macronutrients (10 mM NH4NO3, 9.4 mM KNO3, 0.37 mM MgSO4∙7H20, 0.62 mM KH2PO4 and 1.13 mM CaCl2), micronutrients (50 μM H3BO3, 36.6 μM MnSO4∙ H20, 15 μM ZnSO4∙7H20, 0.57 μM NaMoO4∙2H2O and 0.05 μM CoCl2∙6H20), 50 μM Fe-citrate 0.25 mM KI, 0.05% MES, 1% sucrose and 0.8% phytoagar; pH 5.7-5.8. Seedlings were grown in Cu deficiency (0 μM CuSO4),or Cu excess (10 μM CuSO4).
Growth protocol
Seeds were surface sterilized by treatment with 70% ethanol for 20 min, followed by commercial bleach (2.5 % sodium hypochlorite) containing 0.05 % Triton X-100 for 10 min, and finally, four washes with sterile distilled water. Stratification of the seeds was conducted in the dark at 4ºC for 2 days. Seeds were sowed in ½ MS medium (see specifications in treatment protocol) and grown for 6-7 days under 12 hours of light + 12 hours of darkness photoperiod conditions.
Extracted molecule
total RNA
Extraction protocol
Seedlings were frozen in liquid nitrogen, and grounded in mortars dipped in liquid nitrogen. Total RNA was extracted using the Quiagen Plant RNeasy Mini Kit followong the manufacturer´s recommendations
Label
Cy3
Label protocol
RNA was amplified using the MessageAmp II aRNA Amplification kit (Ambion), basically as described by the manufacturer. 5-(3-aminoallyl)-UTP (aa-UTP) (Ambion) was incorporated into the amplified RNA (aRNA) at ratio 1:1 with UTP. After purification 7.5 µg of aminoallyl-aRNA (aa-aRNA) were used in a coupling reaction with either fluorescent Cy3 or Cy5 as reactive N-hydroxyl succinimidal dyes (NHS-dyes) (Amersham Pharmacia Biotech, Piscataway, NJ). For this, each aliquot of Cy3 or Cy5 were dissolved in 20 µL of DMSO and used in four coupling reactions. aa-aRNA was dissolved in 15 µL of freshly-made 0.1 M NaCO3 pH 9.0, and mixed with 5 µl of either Cy-3 or Cy-5 dye. Coupling was allowed for 1 h at RT in the dark. Reaction was stopped with 35 µL of 100 mM sodium acetate pH 5.2 and incubated 5 min in the dark. Fluorescent aa-aRNA was purified using a MegaClear purification column (Ambion) as described by the manufacturer. Coupled aa-aRNA (250-300 pmol Cy dye in 5-7 µg aa-aRNA) was dried in a speedvac and dissolved in 2.5 µL of filtered water.
ecotype background: Col-0 genotype/variation: wild type tissue: 7 days seedling growth condition: MS withCupper
Treatment protocol
Seeds of Arabidopsis thaliana, ecotype Columbia-0 (Col-0) and the transgenic lines COPT1OE were surface-sterilized and stratified for 2 d at 4°C and were germinated in ½ MS medium plates including 1% sucrose (Murashige, 1962) supplemented or not with the indicated concentrations of cupper. In order to know the effects of both Cu, the components of the ½ MS medium were prepared separately according to the conditions: macronutrients (10 mM NH4NO3, 9.4 mM KNO3, 0.37 mM MgSO4∙7H20, 0.62 mM KH2PO4 and 1.13 mM CaCl2), micronutrients (50 μM H3BO3, 36.6 μM MnSO4∙ H20, 15 μM ZnSO4∙7H20, 0.57 μM NaMoO4∙2H2O and 0.05 μM CoCl2∙6H20), 50 μM Fe-citrate 0.25 mM KI, 0.05% MES, 1% sucrose and 0.8% phytoagar; pH 5.7-5.8. Seedlings were grown in Cu deficiency (0 μM CuSO4),or Cu excess (10 μM CuSO4).
Growth protocol
Seeds were surface sterilized by treatment with 70% ethanol for 20 min, followed by commercial bleach (2.5 % sodium hypochlorite) containing 0.05 % Triton X-100 for 10 min, and finally, four washes with sterile distilled water. Stratification of the seeds was conducted in the dark at 4ºC for 2 days. Seeds were sowed in ½ MS medium (see specifications in treatment protocol) and grown for 6-7 days under 12 hours of light + 12 hours of darkness photoperiod conditions.
Extracted molecule
total RNA
Extraction protocol
Seedlings were frozen in liquid nitrogen, and grounded in mortars dipped in liquid nitrogen. Total RNA was extracted using the Quiagen Plant RNeasy Mini Kit followong the manufacturer´s recommendations
Label
Cy5
Label protocol
RNA was amplified using the MessageAmp II aRNA Amplification kit (Ambion), basically as described by the manufacturer. 5-(3-aminoallyl)-UTP (aa-UTP) (Ambion) was incorporated into the amplified RNA (aRNA) at ratio 1:1 with UTP. After purification 7.5 µg of aminoallyl-aRNA (aa-aRNA) were used in a coupling reaction with either fluorescent Cy3 or Cy5 as reactive N-hydroxyl succinimidal dyes (NHS-dyes) (Amersham Pharmacia Biotech, Piscataway, NJ). For this, each aliquot of Cy3 or Cy5 were dissolved in 20 µL of DMSO and used in four coupling reactions. aa-aRNA was dissolved in 15 µL of freshly-made 0.1 M NaCO3 pH 9.0, and mixed with 5 µl of either Cy-3 or Cy-5 dye. Coupling was allowed for 1 h at RT in the dark. Reaction was stopped with 35 µL of 100 mM sodium acetate pH 5.2 and incubated 5 min in the dark. Fluorescent aa-aRNA was purified using a MegaClear purification column (Ambion) as described by the manufacturer. Coupled aa-aRNA (250-300 pmol Cy dye in 5-7 µg aa-aRNA) was dried in a speedvac and dissolved in 2.5 µL of filtered water.
Hybridization protocol
DNA onto slides was rehydrated by placing the slide upside-down over a water bath at 60°C for 10 s, and snap dry onto a heat block at 65°C. Rehydratation was repeated 3 times. DNA was immobilized by UV light by exposing the slides DNA-side-up to 65 mJ in a UV cross-linker (Stratagene). Slides were then washed twice in 0.1% SDS and 4 times in water, 2 min each wash at RT, and finally dipped in 96% ethanol for 1 min and dried by centrifugation at 2000 rpm 1 min at RT. Slides were prehybridized for 30 min at 42°C with 100 µL of prehybridization solution (6x SSC (Sigma), 1% BSA (Sigma) and 0.5% SDS (Sigma)), under a 60x22 mm coverslip LifterSlip (Erie Scientific) in a microarray hybridization chamber (ArrayIt Hybridization Cassette, Telechem). Slides were rinsed 5 times in H2O in a slide rack at RT and dried by centrifugation at 2000 rpm 1 min at RT. Slides were hybridized immediately. Both coupled aa-aRNA (Cy3 and Cy5) were mixed and fragmented in the presence of 20 µg of tRNA (Ambion) and 20 µg of poly(A) (Roche) using the RNA fragmentation Reagent (Ambion) as described by the manufacturer, in a final volume of 10 µL. Fragmented aa-aRNA was used directly in the hybridization mix, which contains of 50 µL deionized formamide (Sigma), 30 µL 20x SSC, 5 µL 100x Denhardt´s solution (Sigma), and 5 µL 10% SDS in a final volume of 100 µL. Hybridization mixture was denatured at 95 °C for 5 min, spun briefly, and applied by capillary between a pre-treated slide (see above) and a 60x42 mm coverslip LifterSlip. Slides were incubated o/n at 37°C in a microarray hybridization chamber (ArrayIt Hybridization Cassette, Telechem). Slides were washed sequentially once in 1x SSC 0.1% SDS 5 min at 30°C; once in 0.2x SSC 0.1% SDS 5 min at 30°C; twice in 0.1x SSC 2 min each at 30°C; and finally 0.01x SSC 10 sec at RT. Slides were dried by centrifugation at 2000 rpm 1 min at RT.
Scan protocol
Hybridized microarray slides were scanned at 532-nm for the Cy3 and 635-nm for the Cy5 dyes, with a GenePix 4000B scanner (Axon Molecular Devices, Sunnyvale CA), at 5-nm resolution and 100% laser power.
Data processing
Spot intensities were quantified using GenePix Pro 6.0 microarray-analysis software (Axon Molecular Devices, Sunnyvale CA). Spots with a net intensity in both channels lower than the median signal background plus twice standard deviations were removed as low signal spots. Data were normalized by median global intensity using the GenePix Pro 6.0 software. Linear normalized log2 ratio (COPT1 OE/Col-0). Values for COPT1-OE vs Col Cu Def Rep1, COPT1-OE vs Col Cu Exc Rep2 and COPT1-OE vs Col Cu Exc Rep3 have been dye-swap corrected
