Cultivar: Taipai 309 (IRGC accession 42576); Age: 45 d; Tissue: middle section of the blades of fully expanded green leaves from 4 - 5 plants
Biomaterial provider
International Rice Research Institute (IRRI, Manila, Philippines)
Treatment protocol
see growth protocol
Growth protocol
Seeds were pre-germinated in tab water at 28°C for ten days. Then, plantlets were transferred in 10 cm diameter pots filled with 540 g sand mixed with 8 g of Lewatit HD 50 (Lanxess, Langenfeld, Germany), an ion-exchange resin loaded with nutrient ions and 0.4 g Fetrilon Combi (Compo, Münster, Germany). Pots were transferred to a climate chamber with 12 h day length at 600 µE m-2 s-1; temperature was 26°C in the light and 22°C at night, with a relative humidity of 75% in the light and 70% at night. Pots were positioned in polypropylene boxes filled with water to the level of the substrate surface. Pot surfaces were covered with black, pinpricked polythene film (Aquafol, Reinmann, Emsdetten, Germany) to prevent growth of algae. Forty-five days after sowing, plants were harvested five hours after the beginning of the light period. Samples for expression profiling were harvested from the middle section of the blades of fully expanded green leaves and immediately frozen in liquid nitrogen.
Extracted molecule
polyA RNA
Extraction protocol
PolyA+-RNA was extracted with magnetic beads (Dynabeads oligo (dT)25, Dynal, Oslo, Norway) following the manual instruction. In brief, 100mg leaf material was homogenized for 1.5 min at 28 Hz in a ball mill (Retsch) and 1.5 ml Lysis buffer (100 mM Tris, 500 mM LiCl, 10 mM EDTA, 1 % LiDS, 5 mM DTT, pH 8) was added. The solution was mixed and centrifugated at 4°C for 10min at 14000 rpm. 125µl beads were washed twice with 200µl Lysis buffer and were added to the supernatant. After gentle mixing for 5 min beads were washed twice with 1 ml washing buffer A (10 mM Tris, 150 mM LiCl, 1 mM EDTA, 0.1 % LiDS, 0.05% Tween, pH 8), beads were transferred to a new reaction vial and beads were washed twice with washing buffer B (10 mM Tris, 150 mM LiCl, 1 mM EDTA, 0.05% Tween, pH 8). PolyA+-RNA was extracted two times with 10 µl 10 mM Tris pH 7.5 heating 2 min to 95oC. For DNase treatment 20 µl RNA, 4 µl 5x Superscript III Puffer, 0.5 µl RNaseOUT (40 U, Invitrogen), 1 µl DNase (Roche) were incubated for 15 min at room temperature. After adding 1 µl 110 mM EDTA reaction was stopped by incubating for 10 min at 70°C. mRNA from four plants originating from the same experiment, condition and cultivar was pooled. cDNA was synthesized using SuperScript III (Invitrogen, Carlsbad, CA, USA) and purified by precipitation, using Bioline Sure Clean (Bioline, Luckenwalde, Germany).
Label
Alexa Fluor 532
Label protocol
For labeling 1 mg cDNA (in 15 µl buffer (10 mM Tris, pH 8.5)) were mixed with 5 µl of the fluorescent dye (Alexa Fluor 532 and 647 (Qiagen, Hilden, Germany)) and incubated for 30 - 45 min at 85 °C in the dark, followed by adding of 5 µl Stop Solution and an incubation for 10 min at room temperature.
Cultivar: Taipai 309 (IRGC accession 42576); Age: 45 d; Tissue: middle section of the blades of fully expanded green leaves from 4 - 5 plants
Biomaterial provider
International Rice Research Institute (IRRI, Manila, Philippines)
Treatment protocol
see growth protocol
Growth protocol
Seeds were pre-germinated in tab water at 28°C for ten days. Then, plantlets were transferred in 10 cm diameter pots filled with 540 g sand mixed with 8 g of Lewatit HD 50 (Lanxess, Langenfeld, Germany), an ion-exchange resin loaded with nutrient ions and 0.4 g Fetrilon Combi (Compo, Münster, Germany). Pots were transferred to a climate chamber with 12 h day length at 600 µE m-2 s-1; temperature was 26°C in the light and 22°C at night, with a relative humidity of 75% in the light and 70% at night. Pots were positioned in polypropylene boxes filled with water to the level of the substrate surface. Pot surfaces were covered with black, pinpricked polythene film (Aquafol, Reinmann, Emsdetten, Germany) to prevent growth of algae. Twenty-six days after sowing, water was removed from half of the boxes and plants were left to dry for four days, until the soil water content had reached the permanent wilting point (PWP) for 50% of the plants. Thereafter, the soil water content was kept constant to the fixed PWP value over a period of 14 days by weighing each pot at the end of the light period and adding the amount of water lost during the last 24 h. After a total of 19 days of drought stress, plants were harvested five hours after the beginning of the light period. Samples for expression profiling were harvested from the middle section of the blades of fully expanded green leaves and immediately frozen in liquid nitrogen.
Extracted molecule
polyA RNA
Extraction protocol
PolyA+-RNA was extracted with magnetic beads (Dynabeads oligo (dT)25, Dynal, Oslo, Norway) following the manual instruction. In brief, 100mg leaf material was homogenized for 1.5 min at 28 Hz in a ball mill (Retsch) and 1.5 ml Lysis buffer (100 mM Tris, 500 mM LiCl, 10 mM EDTA, 1 % LiDS, 5 mM DTT, pH 8) was added. The solution was mixed and centrifugated at 4°C for 10min at 14000 rpm. 125µl beads were washed twice with 200µl Lysis buffer and were added to the supernatant. After gentle mixing for 5 min beads were washed twice with 1 ml washing buffer A (10 mM Tris, 150 mM LiCl, 1 mM EDTA, 0.1 % LiDS, 0.05% Tween, pH 8), beads were transferred to a new reaction vial and beads were washed twice with washing buffer B (10 mM Tris, 150 mM LiCl, 1 mM EDTA, 0.05% Tween, pH 8). PolyA+-RNA was extracted two times with 10 µl 10 mM Tris pH 7.5 heating 2 min to 95oC. For DNase treatment 20 µl RNA, 4 µl 5x Superscript III Puffer, 0.5 µl RNaseOUT (40 U, Invitrogen), 1 µl DNase (Roche) were incubated for 15 min at room temperature. After adding 1 µl 110 mM EDTA reaction was stopped by incubating for 10 min at 70°C. mRNA from four plants originating from the same experiment, condition and cultivar was pooled. cDNA was synthesized using SuperScript III (Invitrogen, Carlsbad, CA, USA) and purified by precipitation, using Bioline Sure Clean (Bioline, Luckenwalde, Germany).
Label
Alexa Fluor 647
Label protocol
For labeling 1 mg cDNA (in 15 µl buffer (10 mM Tris, pH 8.5)) were mixed with 5 µl of the fluorescent dye (Alexa Fluor 532 and 647 (Qiagen, Hilden, Germany)) and incubated for 30 - 45 min at 85 °C in the dark, followed by adding of 5 µl Stop Solution and an incubation for 10 min at room temperature.
Hybridization protocol
Prehybridization was done following the manufactures instructions: blocking of slide for 20 min at 42°C with Block Solution (2X SSC, 0.05% SDS, 0.25% NaBH4) followed by washing of slide with 1X SSC, 0.2X SSC and rinsing with Nanopure water. For hybridization labelled DNA was dissolved in 130µl hybridization buffer (0.1 % SDS, 25 % Formamide, 5x SSC, 10 mg/ml BSA, 40 mM Na3PO4 (pH 6.8)) and applied to the pre-warmed slide in the hybridization station Hybarray12 (Perkin Elmer, Wellesley, MA, USA). The Initial hybridization temperature was 45°C. The hybridization temperature was stepwise reduced every hour by 1K to reach final hybridization temperature of 41°C. After 10-12 hours of hybridization at 41°C, slides were washed with 2X SSC, 0.2% SDS at 42°C, 2X SSC and 0.2X SSC (all 4 cycles with 20s flow and 40s hold) and very shortly in Nanopure water, all at room temperature. Slides were dried in a centrifuge.
Scan protocol
Microarrays were scanned with a FLA-8000 laser scanner (Fuji, Tokyo, Japan) at maximum intensity. Software version was FLA-8000 V 1.12.
Description
no additional description
Data processing
The software GeneSpotter 2.4.3 (Microdiscovery, Berlin, Germany) was used to fit the grid position of the spots and to calculate spot intensities. Spots for which intensity was affected by dust or dirt were flagged manually (user tag column = 1). Background correction was done based on the expression signal of 218 spots representing a hygromycin resistance gene that is not in the genome of the investigated cultivars. Any spot with an expression below the mean plus threefold standard deviation of the intensity of the hygromycin gene spots of the respective array and dye was labelled as below background (tag column = 1). Normalization was performed using the R package limma (R 2.3.1, limma version 2.7.3; (Smyth, 2005)). The methods Robustspline for within array normalization and Quantile for between array normalization were applied.
