tissue: rice panicle time point: 20min after 40 degree
Treatment protocol
The proper rice plants transplanted into pots were moved in a growth chamber (Binder, Tuttlingen, Germany) under the condition of 32°C with 80% humidity at day, and 28°C with 80% relative humidity at night for one day, and then at 40°C with 80% relative humidity and 600 μmol m-2 s-1 of photosynthetically active radiation for 8hr.
Growth protocol
Heat-tolerant rice cultivar 996 was cultivated in the field until reproductive stage
Extracted molecule
total RNA
Extraction protocol
The rice panicle of post-meiosis was collected at the time point of 0 min (as control), 10min, 20 min, 60min, 2 hr, respectively, after heat treatment, and frozen in liquid nitrogen immediately, and stored at -80°C for microarray hybridization.
Label
Cy3
Label protocol
Cyanine-3 (Cy3) labeled cRNA was prepared from 2μg RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by QIAGEN RNeasy Mini Kit (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
Hybridization protocol
875 ng of Cy3-labelled cRNA was fragmented at 60°C for 30 minutes in a reaction volume of 55μl containing 25x Agilent fragmentation buffer and 10x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 55μl of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Rice Genome Oligo Microarrays (015241) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
Scan protocol
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2565BA) using one color scan setting for 4x44k array slides (Scan Area 61x21.6 mm, Scan resolution 5μm, Dye channel is set to Green and Green PMT is set to 100% and 10%, respectively), and the scan data with 100% and 10% PMT were merged automatically.
Data processing
The above hybridization signal data files in text format from the Feature Extraction Software were imported into GeneSpring GX (Agilent Technologies). The data were marked with specific flag such as P, A, M (representing detected, not detected and compromised, respectively) using the default parameter and algorithm, and then normalized by Quantile algorithm followed by the process of baseline to median of all samples. The normalized data from two replicates of each sample were log2 transformed, and the correlation coefficient of replicates was determined through hierarchical clustering using complete linkage algorithm as implemented TIGR MeV version 4.0.
