2.5 RNA isolation, cDNA labeling and microarray hybridization
For microarray analysis, 30-days old WT and Stpfld 252 plants were subjected to short-term drought treatment as described above. Young fully expanded leaves belonging to the fifth node were collected after three days of water withdrawal in the treated group. Leaf material from 10 different plants of each genotype and treatment were frozen in liquid nitrogen and ground with Mixer Mill MM400 (Retsch). Two pools of biological samples, each from two independent experiments per genotype and condition were used for the microarray analysis. Leaf RNA was extracted according to Logemann et al. (1987). RNA quantity and quality were determined with a NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE) and by visual inspection after electrophoresis, respectively. One microgram of RNA from each sample was treated with RQ1 DNase (Promega, Madison, WI) according to the manufacturer’s instructions, and used as template to generate cDNA with M-MLV Reverse Transcriptase (Promega) and oligo (dT)12−18.
Gene expression profiles were assessed with Potato Oligo Chip Initiative (POCI) microarray, a 60-mer oligo-based 4 × 44k Agilent microarray (AMADID: 015425), consisting of 42034 60-mer probes (Kloostermanet al. 2008). Sample labeling and hybridization were performed as described in the one-color microarray-based gene expression analysis protocol including the one-color RNA spike-in Kit (v5.0.1, Agilent Technologies). Slides were scanned with an Agilent microarray scanner (G2505B) at high resolution. Data were extracted using feature extraction software (v9.5.3, Agilent) by a standard protocol.