ArrayExpressapplication/xmlftp://ftp.ebi.ac.uk/pub/databases/microarray/data/experiment/MEXP/E-MEXP-195/E-MEXP-195.sdrf.txtftp://ftp.ebi.ac.uk/pub/databases/microarray/data/experiment/MEXP/E-MEXP-195/E-MEXP-195.README.txtftp://ftp.ebi.ac.uk/pub/databases/microarray/data/experiment/MEXP/E-MEXP-195/E-MEXP-195.raw.1.zipftp://ftp.ebi.ac.uk/pub/databases/microarray/data/experiment/MEXP/E-MEXP-195/E-MEXP-195.idf.txtprimaryOK20000122TranscriptomicsLemonnier Gaëtan(Make:Axon Instruments,Model:GenePix 4000A)Default scanner softwareArabidopsis Thalianatranscription profiling by arrayhttps://www.ebi.ac.uk/arrayexpress/experiments/E-MEXPfirstname.lastname@example.orgArrayExpressGrowth Protocol - Arabidopsis Col0 Seedlings were cultured on a Murashige and Skoog medium as described by Estelle and Somerville (1987) but without sucrose. Seeds were arrayed individually on a Nylon filter (BioTechnoFix) on 9cm petridishes and incubated for 72h at 4°C, followed by 4h at 20°C, under white light (100µmol/m2s). Plates were wrapped in 3 layers of aluminum foil and incubated at 20°C for the indicated time periods. For the isoxaben treatment, the Nylon filters with the seedlings were transferred under green safe light to a medium supplied with 4nM isoxaben in a final concentration of 5,6mM DMSO or to a control medium containing the same concentration of DMSO. Upon harvest, hypocotyls were dissected out under green light (approximately 30 hypocotyls per experiment), the dissection took approximately 10 min per time point. 16 harvests were pooled, in total 475 hypocotyls, for each time point. RNA was extracted using the trizol reagent (Invitrogen).
Sample Processing - For the isoxaben treatment, the Nylon filters with the seedlings were transferred under green safe light to a medium supplied with 4nM isoxaben in a final concentration of 5,6mM DMSO or to a control medium containing the same concentration of DMSO
Hybridization - Slide preparation In a Coplin jar, prepare 50 ml of pre-hybridization solution : SSC 20 X 12,5 ml BSA 10 % 5 ml SDS 10 % 0,5 ml H2O 32 ml Pre-heat this solution at 42 °C and préhybridize slides at 42 °C for at least 45 mn. Wash the slides in milli-Q water, and then isopropanol. Blow-dry the slides. THE SLIDES MUST BE USED WITHIN 1 HOUR! Hybridization Prepare 2X hybridation mix (50 µl per slide) : SSC 20x 25 µl SDS 10% 1µl Formamide 25 µl Denature the probe 1 mn at 95°C and keep on ice. Add one volume of 2X hybridation mix, mix and centrifuge. Put the slide in the Corning hybridization chamber, and place a cover slip on it. Pipet the probe between the slide and the cover slip. Put 10 µl of water in the two holes and close the chamber. Incubate in a water bath at 42 °C over-night (15h) Washes Soak in 2X SSC, 0,2 % SDS at 42°C to remove the cover slip, then wash in : - 1X SSC, 0,2 % SDS (42°C) 4 min - 0,2X SSC, 0,2 % SDS 4 min - 0,05X SSC 4 min Blow dry the slide or centrifuge for 2 min at 600 rpm. Store in a dry and dark place until scanning.
Labeling - 5 µg of aRNA (8µl) mixed with 2 µl of random nonamers 1µg/µl and 0.5 µl of RNAse Out 40 U/µl, denatured 10 min at 70 °C and chilled on ice. The followig components were added to the sample: 4 µl of first strand buffer 5 X, 1 µl of 10 mM dNTP/2mM dCTP, 2 µl of 0.1 M DTT, 1.5 µl of Cy3-dCTP (Amersham Pharmacia Biotech, 25nmol tube, PA5502), 1 µl of SuperScript II RT 200U/µl. Then it was incubated at 42°C for 2.5 hours and chilled on ice. The sample was denaturated by adding 2 µl of NaOH 2.5M and incubated at 37 °C for exactly 15 min, then was added 10 µl of 2M MOPS and put on ice. The dyes were purified with the QIAquick PCR Purification Kit (QIAGEN).
Nucleic Acid Extraction - Plant tissue was ground to fine powder using liquid nitrogen in a 2mL eppendorf tube. The samples were homogenized in TRIzol Reagent (Invitrogen) : 1 mL for 50-100 mg of plant tissue, by vortexing and incubated at room temperature for 5 min. Chloroform (200µl/1mL TRIzol) was added and the capped tubes were hand-shaken for 15 sec. They were incubated for 2-3 min, then centrifuged for 15 min at 11000 rpm at 4°C. The aqueous phase was transferred to a new 1.5 mL tube and 500ul of isopropanol was added. The samples were incubated for 10 min at room temperature then centrifuged for 10 min at 11000 rpm at 4°C. The supernatant was removed and the pellet was washed with 1 mL of 75% ethanol in DEPC-treated water. The samples were centrifuged for 5 min at 11000 rpm at 4°C. The pellet was air-dried for 10 min at room-temperature, then resuspended in 500 µl DEPC-treated water, and precipated with 2 volumes of 100 % ethanol and 1/10 V of Sodium acetate 3M, pH5,2. The samples were precipitated 30 min at -20 °C and centrifuged for 15 min at 10000 rpm at 4°C. The supernatant was removed, the pellet was air-dried and resuspended in DEPC-treated water. The RNA solution was purified on Qiagen RN-Easy column. The RNA quality was then acessed on Agilent Bioanalyser following the manufacturer's protocol and the quantity is determined by Ribogreen.Assay Data Transformation - Analysis of 2 technical repeats The statistical analysis was based on two dye-swaps, i.e. four arrays each containing the 24576 GSTs and 384 controls. The controls were used for assessing the quality of the hybridizations but were not included in the statistical tests or the graphical representation of the results shown in the Results. For each array, the raw data comprised the logarithm of median feature pixel intensity at wavelength 635 nm (red) and 532 nm (green). No background was subtracted. In the following description, log-ratio refers to the differential expression between leaves and flowers. It is either log2(red/green) or log2(green/red) according to the experiment design. An array-by-array normalization was performed to remove systematic biases. First, we excluded spots that were considered badly formed features by the experimenter. Then we performed a global intensity-dependent normalization using the loess procedure (see (Yang et al., 2002) to correct the dye bias. Finally, on each block, the log-ratio median is subtracted from each value of the log-ratio of the block to correct a print-tip effect on each metablock. To determine differentially expressed genes, we performed a paired t-test on the log-ratios. The number of observations per spot varies between 2 and 4 and is inadequate for calculating a gene-specific variance. For this reason we assume that the variance of the log-ratios is the same for all genes and we excluded 256 spots displaying extremes of variance (too small or too large). The raw P-values were adjusted by the Bonferroni method, which controls the Family Wise Error Rate (FWER)(Ge et al., 2003).