arabidopsis.org/), and the UniProt Knowledgebase (http://www.ebi.ac.uk/).
Functional annotations were then assigned based on OSI-744 in vivo sequence similarity (with E-value of 10− 5) with manual adjustment when necessary. All of the probes were grouped into functional categories and metabolic pathways based on the Mips Functional Catalogue (http://mips.gsf.de/) and KEGG ,  and . Adhering to the established method  and , we identified statistically enriched KEGG pathways and gene families of transcription factors in differentially expressed genes based on a background distribution from the whole chip. The expression levels of genes were measured by detection calls and signal intensities using Micro Array Suite 5.0 software with a target signal of 100. All pair-wise differentially expressed genes were identified using SAM software (http://www-stat.stanford.edu/~tibs/SAM/) to analyze data from all remaining maize probe sets. A false discovery rate parameter of 1% was set for the SAM analysis. Genes that were called absent more than twice among the three replicates in both the control and treatment arrays were then considered to be not unexpressed under both conditions and were excluded from the list. A 5 μg sample of total RNA was used for cDNA synthesis using the Invitrogen Reverse Transcription Reagents
Kit (Invitrogen). Gene-specific primers were designed using Primer Express 2.0 software (Applied Biosystems) and synthesized by Sangon Biotech Co. Ltd. (Shanghai). Relative quantitative analysis was performed using an Applied Biosystems 7900HT real-time PCR system (Applied Biosystems) under the Proteases inhibitor following conditions: 94 °C for 3 min (1 cycle); 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s (40 cycles). Transcript abundance was identified using SYBR
Green PCR Master Mix (Applied Biosystems). Each reaction contained 1 × buffer, 0.25 μmol L− 1 of each primer, and approximately 2 ng of cDNA in a final volume of 20 μL. Three replicates were employed for each tested sample and template-free negative control. Mitochondrial 5S RNA was used as an internal control to normalize all data. Melting curves were performed on the product to test whether only a single product was amplified without primer-dimers and other bands. The resultant products with Arachidonate 15-lipoxygenase all primer combinations were initially visualized on 2% agarose gels to confirm the generation of a single product of the correct size. To identify miRNAs in developing kernel of a maize viviparous mutant at different developmental stages, RNAs of 18 to 26 nt in length were purified and cloned from small RNAs (~ 200 nt) isolated from germinating maize kernels for subsequent sequence analysis. Approximately 64 concatamer clones were sequenced to generate 540 sequences after discarding low-quality and self-ligated linker sequences. Of these, 56 small RNA-cDNA sequences were outside the expected range of nucleotide lengths (18–26 nt).