Affymetrix Generic Hybridization
nucleic acid labeling protocol
Labeling of samples was carried out according to the manufacturer’s standard protocol
Biotinylated target cRNA was prepared from 4 µg of total RNA following the manufacturer’s specifications (Affymetrix). The samples were then hybridized to Affymetrix GeneChip(r) Rice Genome Arrays, which contain probes to query ~51K transcripts representing both the Japonica and Indica cultivars. The chips were thereafter washed and stained in a GeneChip(r) Fluidics Station 450. Scanning was carried out with GeneChip(r) Scanner 3000 and image analysis was performed using GeneChip(r) Operating Software. (Parameters: Scanning hardware = GeneChip Scanner 3000 [Affymetrix], Scanning software = Scanning software)
Seeds of two rice cultivars, Oryza sativa, ssp. Japonica, cv. Jumli Marshi (JM) and ssp. Indica, cv. IR64 (IR64), were first soaked in water for 16 hours at room temperature and thereafter grown on standard soil in 14 hours photoperiod, with a day/night air temperature of 25 degrees C/20 degrees C and 250 µmol m2 s-1 light. (Parameters: time unit = seconds, temperature unit = C)
At mid-day, three week old plants were transferred to growth chambers (Percival) in the same photoperiod, but with an air temperature of 4 degrees C and a light intensity of 100 umol m2 s-1. Pooled leaf tissue from five individual Jumli Marshi plants was harvested at 0, 0.5, 2, 4, 8, and 24 hours, frozen in liquid nitrogen and stored at -80 degrees C until further analysis.
Total RNA was extracted from JM leaf tissue with TRIZOL reagents (Invitrogen) according to the manufacturer’s protocol and purified by RNeasy MinElute Cleanup Kit (Qiagen). (Parameters: Extracted product = total_RNA, Amplification = none)