ecotype: Landsberg erecta (Ler) Genotype: flu1-1 Plant material : rosette leaves of 3 week-old plants grown under continuous light 90 mmol. m-2 . s-1, before bolting
Treatment protocol
Plants were sprayed either with a solution of 20 microM paraquat (methyl viologen, Sigma) in 0.1% Tween or with Tween alone, and rosette leaves were harvested at 1, 2, and 4 h after spraying.
Growth protocol
Plants were grown on soil for 3 weeks under continuous light at 90 mmol. m-2 . s-1
Extracted molecule
total RNA
Extraction protocol
total RNA extraction was performed according to Melzer, S., Majewski, D. M. & Apel, K. (1990) Plant Cell 2, 953-961: RNA was isolated with some modifications as described by Chirgwin et al. (1979). The plant material was ground under liquid nitrogen with a mortar and a pestle and homogenized in 4 mL of lysis buffer (4 M guanidinium isothiocyanate, 50 mM Hepes-KOH, pH 7.4, 2% Nlauroyl-sarcosin and 1 YO o-mercaptoethanol) with an Ultra Turrax (Janke and Kunkel, D7813 Stanfen, F. R. G.). Cell debris and denatured proteins were pelleted and the nucleic acids in the supernatant were precipitated overnight in the presence of 0.75 volumes of ethanol. The pellet was homogenized in 4 mL of Tes (10 mM Tris-HCI, pH 7.5, 1 mM EDTA, and 0.1% SDS) with the help of a potter, and the homogenate was extracted with phenol/chloroform and chloroform. Total RNA was precipitated overnight with 2 M LiCl at 4°C. Poly(A)-containing RNA was isolated according to Aviv and Leder (1972).
Label
biotin
Label protocol
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 20 ug total RNA (Eukaryotic Target Preparation, 2002, Affymetrix).
Hybridization protocol
Following fragmentation, 15 ug of cRNA were hybridized for 16 hr at 45C on GeneChip Arabidopsis ATH1 Genome Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 400, protocol EukGE-WS2v4.
Scan protocol
GeneChips were scanned using the Agilent GeneArray Scanner
Description
Gene expression data from rosette leaves of 3-week-old flu mutant plants grown under continuous light, 2h after mock treatment with tween 0.1%
Data processing
The data were analyzed with Microarray Suite version 5.0 (MAS 5.0) using Affymetrix default analysis settings and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 100.