J Appl Genetics (2011) 52:161–169 phenol-chloroform-isoamylalcohol (25:24:1) and an RNeasy mini plant kit (Qiagen). The quantity of total RNA was determined by measuring absorbance at 260 nm and 280 nm by using a Nanodrop ND-1000 spectropho- tometer (Thermo Fisher Scientific Inc., Wilmington, NC, USA). In addition, the level of protein contamination in the RNA was determined based on the A260/A280 ratio. Only RNA samples with ratios of 2.0-2.2 were used for these experiments. Anthocyanin analysis The aleurone layer, about 10% of the outer layer of black rice, was extracted with 10 mL of 1% HCL in methanol for 24 h in the dark. Quantification of anthocyanin was performed using a Waters CapLC XE pump (Millford, MA, USA) with Waters 2996 photodiode array detection system. A Symmetry C18, 5 μm (0.32× 150 mm) column, operated at 25°C, from Waters was used for separation. The mobile phase consisted of 0.1% trifluoroacetic acid (TFA) in water (eluant A) and 0.1% TFA in 95% acetonitrile (eluant B). The injection volume for all samples was 0.2 μl. Spectra were recorded from 200 to 600 nm at a flow-rate of 0.5 μl min-1. The electrospray ionization mass spectrometry (ESI-MS) was carried out with a Micromass electrospray interface ZMD 4000 (Micromass, Manchester, UK). Nitro- gen was used as the nebulizing gas. Source blocks and desolvation temperatures were 110°C and 20°C, respec- tively. Prior to analysis, all samples were filtered through a 0.45-μm membrane filter. The standard anthocyanins (i.e., cyanidin-3-O-glucoside and peonidin-3-O-glucoside) were obtained from Extrasynthese (R&D Chemicals, Genay, France), and apigenin, kaempferol and quercetin were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Microarray analysis Microarray analysis was performed using newly designed 135 K Oryza sativa microarrays. These microarrays contained probes to assess 31,439 genes deposited at the International Rice Genome Sequencing Project (IRGSP, http://rgp.dna.affrc.go.jp/E/IRGSP/) and the Rice Annota- tion Project version 2 (RAP2, http://rapdb.dna.affrc.go.jp/). Four 60-nt probes were designed from each gene starting at 60 bp upstream of the stop codon and with a 30-bp shift such that four probes could be used to cover a 150-bp region at the 3' end of the gene. In addition, 200 genes were included from 123 chloroplasts, 74 mitochondria, and 3 selection markers. Therefore, the microarray had a total of 125,956 probes. The microarray was scanned using the GenePix 4000B (Molecular Devices, Inc., Sunnyvale, CA, USA), and signals were digitized and analyzed by 163 Nimblescan (Nimblegen, Madison, WI, USA). To improve the sensitivity and reproducibility of microarray analysis (Irizarry et al. 2003), normalization was processed using the cubic spline normalization method (Workman et al. 2002), and probe-level summarization was processed by Robust Multichip Analysis (RMA) using a median polish algorithm (Sadlier et al. 2004). RT-PCR analysis Reverse transcription-polymerase chain reaction (RT-PCR) analysis was carried out to verify the microarray hybrid- ization data. To reduce experimental variation, a total of 27 RNA samples were extracted from the Dongjin, Heuginju, and Heugseol cultivars at 7, 14, and 21 days after the heading stage, in triplicate. Each RNA was independently isolated for RT-PCR, apart from those used for the micro- array analysis. After treatment with RNase-free DNase followed by phenol extraction and ethanol precipitation, 5 μg of total RNA from each sample was used to synthesize each pool of cDNA using the SuperScript III First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA). For PCR ampli- fication, 1 μl of the resulting cDNA reaction was used as a template. The PCR reactions were carried out in 50-μl volumes with 1.25 unit of Taq DNA polymerase and 20 pmol of each primer pair. The primers were designed based on sequence information available from the Rice Annota- tion Project Database (RAP, http://rapdb.dna.affrc.go.jp) and the NCBI/Primer-BLAST (http:// www.ncbi.nlm.nih. gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome). The PCR program was as follows: 3 min at 94°C, 24 cycles of 30 sec at 94°C, 30 sec at the optimal annealing temperature (58 to 65°C), and 1 min at 72°C, followed by 5 min at 72°C. To validate our RT-PCR results, we performed each experiment three times. The PCR reaction with 24 cycles of amplification was analyzed by gel electrophoresis. Actin mRNA was used as a loading control. Data collection and statistical analysis All experiments were performed using only Cy3 to eliminate the dye-swap error value. Data-based background subtraction using a local background estimator was per- formed to improve fold change estimates on arrays with high background. Spot intensity was calculated as the median value of the spot compared to the background median value. The functional classification categories assessed during COGs analysis using the NCBI/COGs database (http://www.ncbi.nlm.nih.gov/COG/). To deter- mine the processes affected significantly by anthocyanin production, GO analysis was performed using GoMiner