tissue: leaf, vegetative stage cultivar: IR57311 condition: control conditions (28/21°C)
Treatment protocol
Twenty-five days after sowing (DAS) at the 3–4 leaf stage, the temperature settings in the chamber were either kept at the control conditions described above or changed to HNT chamber conditions of 28°C in the light and 28°C at night for 23 days. RH and light regime remained unchanged.
Growth protocol
Plants were grown under control or HNT conditions in a controlled climate chamber. Seeds were pre-germinated in tap water for 12 days, 7 days in the dark at 28°C and the following 5 days under a 12 h light regime at 28°C in the light and 21°C at night. Single seedlings were planted into 22 cm deep 10 cm square pots (TO 10 D, Kauseck, Mittenwalde, Germany) with 540 g potting mixture (two parts pot soil substrate (70% white moss peat, 30% clay), one part sand) mixed with 1 g of slow-release fertiliser (Plantacote Depot 4M; Lanxess, Langenfeld, Germany) and 0.1 g Fetrilon Combi (Compo, Münster, Germany). Plants were grown in a climate chamber (Johnson Controls, York Refrigeration, USA) with 12 h day-length at a photon flux density of 700 µE m–2 s–1 and a lamp distance of 180 cm above the upper box edge (lamps from Iwasaki Eye MT 400 DL/BH E40, DHL Licht, Wülfrath, Germany) at chamber settings of 26°C in the light and 22°C at night, at a RH of 70%. Fifteen pots were positioned together in one polypropylene box (height of 24 cm) filled with 26°C warm tap water. Water was filled to 2 cm below soil surface for 14 days after sowing and then filled above the level of the soil surface till the end of the experiment.
Extracted molecule
total RNA
Extraction protocol
RNA isolation was done from pooled samples of five replicates for each cultivar and for each independent experiment resulting in 36 samples (6 cultivars, 3 HNT and 3 control experiments). Frozen leaf material was homogenized in a ball mill and RNA was extracted from 100 mg leaf material using the protocol described above (paragraph 2.2.5.1). Purified RNA was treated with Turbo DNase-freeTM kit. The integrity of the final RNA samples was checked by denaturing gel electrophoresis on 1.4% (w/v) formaldehyde agarose gels and concentration was determined photometrically (NanoDrop ND-1000 UV-Visspectrophotometer, Nanodrop Technologies, Wilmington, DE). 10 μl of sample (1 to 4 μg RNA) was handed over to the company imaGenes (Berlin, Germany) which performed quality checks, cRNA synthesis and labeling, hybridization and scanning and transfered the raw data back for further analysis.
Label
Cy3
Label protocol
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.2 ug RNA using the Low Input Quick Amp Labeling (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
Hybridization protocol
1.65 ug of Cy3-labelled cRNA (specific activity >6.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 55 µl containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 55 ml of 2x 2x GEx Hybridization Buffer HI-RPM was added to the fragmentation mixture and hybridized to a custom Gene Expression Agilent Microarray (G2514F; DesignID 031554) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
Scan protocol
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2565CA) using one color scan setting for AgilentHD_GX for 4x44k array slides (Scan Area 61x21.6 mm, Scan resolution 5µm, Dye channel is set to Green and Green PMT is set to 100%, Tiff 20bit).
Description
IR57311 in leaves under control conditions (28/21°C) leave_48DAS_28/21°C_replicate3
Data processing
Data normalization was performed by Babelomics software (http://babelomics.bioinfo.cipf.es/), using Agilent one-channel normalization by choosing “substract” normalization for within array normalization. Replicates of probes showed similar expression and were averaged, control samples and quality checks were removed after normalization. Further sequences with only predicted location and function were included in array design but were removed because of missing information about quality of prediction. Also probes for microRNAs were excluded from this study. Replicates, which were found to be outliers, were removed from further analysis, which are IR6226 under control conditions (2 replicates) and Nipponbare under control conditions (1 replicate).
