reference: control time point: Day3 replicate: 3 cultivar: Cv. PS3 tissue: Leaves
Treatment protocol
Salt treatment was started at day 0 by watering the plants with 75 mL of 150 mM NaCl solution while the control plants were watered with the same amount of tap water.
Growth protocol
After in vitro multiplication, plantlets of a Solanum tuberosum Cv. PS3 were transferred for acclimatization in a 13 cm diameter pot with 0.25 dm3 of 3:1 soil:sand mixture during 2 months before salt exposure. The culture room parameters were set at 21°C/18°C (day/night), 12 hours daylight and a light intensity of 40 μmol.m-2.s-1.
Extracted molecule
total RNA
Extraction protocol
The first and second leaves were sampled after 1 and 3 days on two different sets of plants. Leaves were immediately frozen in liquid nitrogen before being stored at -80°C. For each treatment and time-point, three biological replicates were sampled. Total RNA was extracted from 100 mg of frozen leaves with the RNeasy Plant Mini Kit (Qiagen, Leusden, The Netherlands) including DNase treatment, following the manufacturer’s instructions. Quality control was performed with the RNA Nano assay using a 2100 Bioanalyzer (Agilent Technologies, Diegem, Belgium). RNA samples with a RIN (RNA integrity number adapted to plant rRNA profiles) lower than 7 were excluded from the experiment. RNA purity and concentration were measured by the absorbance at 260 nm and 280 nm using a DU800 spectrophotometer (Beckman Coulter, Villepinte, France).
Label
Cy5
Label protocol
Labelling of 15 µg RNA per sample was performed following the TIGR protocol. Reverse transcription was performed using Superscript II kit (Invitrogen) during 2 hours (see supplemental data). The reaction was stopped by addition of 10 µL 0.5 M EDTA. Untranscribed RNA was degraded by addition of 10 µL 1 M NaOH and incubation at 65°C during 15 min. Sixty microliters of 1 M Tris-HCl were added for neutralisation. aa-cDNA purification was performed using the QIAquick PCR purification kit (Qiagen, Leusden, The Netherlands). Washing steps were performed using 500 µL of 5 mM KPO4 pH=8.5, 80% EtOH solution and elution was done using two times 30 µL of 4 mM KPO4 pH=8.5 (final volume 60 µL). Precipitation of aa-cDNA was performed (see supplemental data for details) during 1 hour at 80°C. After centrifugation at 16000×g during 20 min, the pellet was washed using cold 75% EtOH and vacuum-dried for 10 min. The pellet was solubilised in 4.5 µL daily-prepared 0.1 M Na2CO3. A volume of 4.5 µL of dye (previously resuspended in DMSO following manufacturer instructions) was then added and the labelling reaction took place during 1 h at room temperature. To remove uncoupled dye, the QIAquick PCR purification kit was used and the elution was performed twice with supplied elution buffer EB (final volume of 65 µL). Cyanine incorporation was controlled by measuring the absorbance at 260 nm (cDNA), 550 nm (Cy3) and 650 nm (Cy5), followed by an adjustment of the Cy3- and Cy5-coupled cDNA quantities.
agent: Salt time point: Day3 replicate: 3 cultivar: Cv. PS3 tissue: Leaves
Treatment protocol
Salt treatment was started at day 0 by watering the plants with 75 mL of 150 mM NaCl solution while the control plants were watered with the same amount of tap water.
Growth protocol
After in vitro multiplication, plantlets of a Solanum tuberosum Cv. PS3 were transferred for acclimatization in a 13 cm diameter pot with 0.25 dm3 of 3:1 soil:sand mixture during 2 months before salt exposure. The culture room parameters were set at 21°C/18°C (day/night), 12 hours daylight and a light intensity of 40 μmol.m-2.s-1.
Extracted molecule
total RNA
Extraction protocol
The first and second leaves were sampled after 1 and 3 days on two different sets of plants. Leaves were immediately frozen in liquid nitrogen before being stored at -80°C. For each treatment and time-point, three biological replicates were sampled. Total RNA was extracted from 100 mg of frozen leaves with the RNeasy Plant Mini Kit (Qiagen, Leusden, The Netherlands) including DNase treatment, following the manufacturer’s instructions. Quality control was performed with the RNA Nano assay using a 2100 Bioanalyzer (Agilent Technologies, Diegem, Belgium). RNA samples with a RIN (RNA integrity number adapted to plant rRNA profiles) lower than 7 were excluded from the experiment. RNA purity and concentration were measured by the absorbance at 260 nm and 280 nm using a DU800 spectrophotometer (Beckman Coulter, Villepinte, France).
Label
Cy3
Label protocol
Labelling of 15 µg RNA per sample was performed following the TIGR protocol. Reverse transcription was performed using Superscript II kit (Invitrogen) during 2 hours (see supplemental data). The reaction was stopped by addition of 10 µL 0.5 M EDTA. Untranscribed RNA was degraded by addition of 10 µL 1 M NaOH and incubation at 65°C during 15 min. Sixty microliters of 1 M Tris-HCl were added for neutralisation. aa-cDNA purification was performed using the QIAquick PCR purification kit (Qiagen, Leusden, The Netherlands). Washing steps were performed using 500 µL of 5 mM KPO4 pH=8.5, 80% EtOH solution and elution was done using two times 30 µL of 4 mM KPO4 pH=8.5 (final volume 60 µL). Precipitation of aa-cDNA was performed (see supplemental data for details) during 1 hour at 80°C. After centrifugation at 16000×g during 20 min, the pellet was washed using cold 75% EtOH and vacuum-dried for 10 min. The pellet was solubilised in 4.5 µL daily-prepared 0.1 M Na2CO3. A volume of 4.5 µL of dye (previously resuspended in DMSO following manufacturer instructions) was then added and the labelling reaction took place during 1 h at room temperature. To remove uncoupled dye, the QIAquick PCR purification kit was used and the elution was performed twice with supplied elution buffer EB (final volume of 65 µL). Cyanine incorporation was controlled by measuring the absorbance at 260 nm (cDNA), 550 nm (Cy3) and 650 nm (Cy5), followed by an adjustment of the Cy3- and Cy5-coupled cDNA quantities.
Hybridization protocol
Probe hybridization was performed according to the TIGR protocol. Probes were precipitated during 1 h at 80°C. After centrifugation at 16000×g at 4°C during 20 min, the probes were washed with cold 75% EtOH, dried, resuspended in 60 µL hybridisation buffer (5X SSC, 50% formamide, 0.1% SDS) and finally denatured at 95°C for 3 min. Hybridisation in individual chambers was done at 42°C during 16 h. Cy5-labelled cDNAs of salt-treated plants were hybridized versus Cy3-labelled cDNAs of control plants. Two slides were performed with a dye-swap (inverted labelling). Slides were washed (see supplemental data) and dried by centrifugation at 30×g during 5 min. The TIGR 10K potato microarrays containing 15,264 cDNAs repeated twice (http://www.jcvi.org/potato/sol_ma_microarrays.shtml) were used for the experiments.
Scan protocol
Slides were scanned using a Professional 4200A microarray scanner (Molecular Devices Corporation, Union City, CA, USA). Two laser channels were used (532 nm and 635 nm) at different settings for the photomultiplier tube (PMT gain). The two PMT gains were automatically adjusted with the Genepix® Pro 6.0 software (Molecular Devices Corporation, Union City, CA, USA) to reach a global intensity ratio close to 1 with pixel saturation equal to 0.001%. Spots with a diameter smaller than 60 µm, spots with a signal intensity below background intensity plus two standard deviations and finally clones annotated “Do not use” were excluded from further analysis. MA plots (log ratio (stress/control) versus mean (log(stress),log(control))) were performed to check the quality of the arrays.
Description
Potato_leaves_control_Vs_salt-Treated_Day3_Rep3
Data processing
To analyse gpr files generated by Genepix® Pro 6.0 software, data were imported into Acuity 4.0 software (Molecular Devices Corporation, Union City, CA, USA) and normalised with the print-tip Lowess method. A “one sample t-test” was first performed for the three slides of each time-point.In order to retain spots with repeatable results, only probes with a p-value below 0.05 were selected. As a second step, probes with a log ratio below -0.5 and above 0.5 were selected to generate the final list.
