7 protocols
Biotin-labeled cRNA samples were mixed in 300 mL of Hybridization Mix (Affymetrix; 900720) containing Hybridization Controls and Control Oligonucleotide B2 (Affymetrix; 900454). Samples were hybridized onto Affymetrix AGRONOMICS1 Arabidopsis tiling arrays and ATH1 arrays for 16 h at 45 C. Arrays were then washed using an Affymetrix Fluidics Station 450 using the FS450_0004 protocol..
Background correction, normalisation, and calculation of probe set summaries were done using RMA (Irizarry et al., 2003) implemented in the Aroma.Affymetrix package (Bengtsson et al., 2008).
Data were log2-transformed by RMA.
The arrays were scanned using an Affymetrix 3000 7G confocal scanner.
Three successive experiments were carried out using seeds of the Arabidopsis thaliana accession Col-0 and osd1-1. For each experiment, seeds of about 20 siliques were harvested. Plants were grown under long-day conditions (16 h light and 8 h darkness) at 18 C.
RNA was amplified and labelled with the GeneChip® IVT Express Labelling kit (Affymetrix, Santa Clara, CA).
Seeds were harvested into 40 ul RNAlater (Sigma, Buchs, Switzerland) . Glass beads (1.25-1.55 mm) were added, and the samples were ground unfrozen in a Silamat S5 (Ivoclar Vivadent, Ellwangen, Germany). RNA was extracted using the RNAqueous kit (Ambion/Applied Biosystems, Lincoln, CA) combined with Plant RNA Isolation Aid (Ambion/Applied Biosystems) according to manufacturer’s instructions. The RNA was subjected to a DNase treatment.