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.