Rice 4 44 k microarray rap db

Total RNA was extracted from the collected samples using a RNeasy Mini Kit (Qiagen, Hilden, Germany), and labeling was performed using Quick Amp Labeling Kit, One-Color (Agilent Technologies) in the presence of cyanine-3 (Cy3)-CTP according to the manufacturer’s protocol. The resulting Cy3-labeled cRNA was purified using a RNeasy Mini Kit (Qiagen, Hilden, Germany). A total of 1000 ng Cy3-labeled cRNA was fragmented and hybridized onto a slide of the rice 4 × 44 K microarray RAP-DB (G2519F#15241; Agilent Technologies). After washing, the slides were scanned on an Agilent G2505B DNA microarray scanner, and background correction of the raw Cy3 signals was performed using Feature Extraction 10.5.1.1 software (Agilent Technologies).

Twelve rootless shoots per bottle were precultured for 4 days in 100ml of LSCS medium to form roots, after which 1mM KNO₂ was added. After 12h, whole roots were collected from 10 of the 12 treated plants per bottle and were immediately frozen in liquid nitrogen and homogenized using Micro Smash (Tomy Seiko, Tokyo, Japan). Three sets of preparations were repeated for each experimental condition. Total RNA was isolated using an RNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA). Microarray experiments were performed using Rice 4×44 K Microarray RAP-DB (G2519F#15241; Agilent, Santa Clara, CA, USA) as described in Takehisa et al. (2012) (link).
The processed raw signal intensity of all probes (45,151) was subjected to 75th-percentile normalization with Subio platform software with the basic plugin (Subio, Kagoshima, Japan) and transformed to a log₂ scale. A total of 24235 probes (corresponding to 17003 genes) were extracted from 12 microarray data sets after ANOVA normalization. Fold change was calculated as the ratio of log₂-transformed signal intensity between the untreated NT control and the treatment. We used 1.0 and −1.0 as the critical points for declaring upregulation and downregulation, respectively. Boxplot analysis was performed using Origin 8.6 software (OriginLab, Northampton, MA, USA).