Raw intensity data ratios were normalized using printtip loess normalization and scaling with the R package Limma.
Slides were scanned using the ScanArray Express HT scanner and the ScanArray v3.0 software (Perkin Elmer). Features were extracted using Imagene 6.0 software.
Slides were denatured in boiling water for 3 min and pre-hybridised for 15 min at 55C with pre-hyb solution (25%formamide, 5x SSC, 0.1%SDS, 1%BSA, filtered through 0.2 um). The probe mix of 1000ng cDNA for both the cy3- and the cy5-labeled sample in 40 ul was added to 40 ul of hybridization solution (50%formamide, 10x SSC, 0.2%SDS, filtered through 0.2 um, + 5 ul herring sperm (1mg/ml)) and denatured for 5 min 95 C. Arrays were hybridized o/n at 42C and washed with SSC and SDS solutions.
All plants were treated with 3 hours of neutral shade (ca 10% of growth conditions) 21 days after transfer to soil (24 days after germination). The three most responsive leaves (petiole and lamina) per plant were harvested and pooled per genotype for RNA isolation and transcription profiling.
Arabidopsis thaliana Ler x Cvi recombinant inbred lines were sown on wet filter paper, stratified (4C) for 3 days and then transferred to a growth chamber (9 h. light (200 umol / m2 / s, 20C, 70% RH), 15 h dark) where they were transferred to individual pots with soil after 3 days and treated after 24 days of growth.
Total RNA was isolated from homogenized material using the RNeasy plant mini kit (Qiagen) following the company’s instructions.
Total RNA was amplified with the MessageAmp aRNA kit (Ambion). Amplified mRNA (5 ug) was used as a template to synthesize modified cDNA with SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA) and random nonamers (Gene Link, Westchester County, NY, USA) for 2 h at 42C with the incorporation of 5-(3-aminoallyl)-dUTP (Ambion; ratio dUTP/dTPP of 7/3), yielding 1-2 ug cDNA. RNA template was removed by hydrolysis with 3 ul 2.5 m NaOH per 30 ul for 15 min at 37C. The hydrolysis was stopped by neutralization with 15 ul 2 m MOPS and put on ice. cDNA was purified using the MineLute PCR purification kit (Qiagen) and labelled with Cy3 or Cy5 mono-reactive dye (Amersham, Buckinghamshire, UK). The reaction was quenched after 60 min using 4.5 ul 4 m hydroxylamine (Sigma-Aldrich) and incubated in the dark for 15 min. The labelled cDNA was purified as described above, and incorporation of Cy3 or Cy5 was determined using a UVmini-1240 spectrophotometer at 550 or 650 nm, respectively.