Arabidopsis ath1 genome array

Microarray experiments were conducted using Affymetrix Arabidopsis ATH1 Genome Array. Experiments were performed as three biological repeats using cDNAs prepared independently from three individual homozygous lines of MeGRXC15 overexpression Arabidopsis that were phenotypic analyzed in plant growth. The transgenic Arabidopsis plants that carried the pG1300 empty vector were used as controls. The experiments and data analysis were performed under the instruction of Affymetrix. Total microarray data were deposited in the NCBI GEO database with the accession number: GSE81136 (MeGRX232-OE). Gene ontology (GO) analyses for significant enrichments of various categories (Additional file 2: Table S5) were performed using MAS 3.0 (http://bioinfo.capitalbio.com/mas3/). The Venn diagrams were created by online tool (http://bioinformatics.psb.ugent.be/webtools/Venn/).

The Affymetrix gene chip Arabidopsis ATH1 Genome Array was used for microarray analysis. Total RNA sample were prepared as described above. All RNA samples were quality assessed by using the Agilent Bioanalyzer 2100 (Agilent, Santa Clara, USA). Complementary RNA synthesis was performed by use of the GeneChip One-Cycle Target Labeling Kit (Affymetrix, Santa Clara, USA). Hybridization, washing, staining, and scanning procedures were performed as described in the Affymetrix technical manual.
Gene expression data were imported directly into GeneSpring (version 11.5, Agilent, Santa Clara, USA). The software was used to normalize the data per chip to the 50^th percentile and per gene to the control samples. Genes that were flagged as absent in two replicates were not considered in the analysis. P values for the Benjamini and Hochberg method (false discovery rates; FDRs) were calculated by GeneSpring. Transcripts were defined as differentially expressed that showed delta signal changes larger than the mean expression value of the whole data set or 2-fold changes with P < 0.05.

Liquid N₂ ground tissue was incubated with Trizol (1 mL per 100 mg of tissue; Invitrogen) at room temperature for 2 min. Chloroform (200 μL) was added, samples gently mixed and centrifuged (12,000 × g, 4°C, 15 min). The aqueous upper phase was retained, and combined with 250 μL high salt solution (0.8 M sodium citrate, 1.2 M sodium chloride) and 250 μL isopropanol. After mixing and centrifuging the samples, the pellet was isolated and washed with ethanol. Samples were then DNase treated (Promega), and a final clean up step was performed using the RNeasy kit (Qiagen), according to the manufacturers protocol. RNA concentration and quality was measured using a BioPhotometer plus (Eppendorf) as well as by agarose gel electrophoresis. Samples were then sent to the Nottingham Arabidopsis Stock Centre (NASC; http://affymetrix.arabidopsis.info/) for analysis. Quality control was first performed using an Agilent Bioanalyzer to check the integrity of the RNA. Following preparation of cDNA according to NASC standard protocols, the samples were hybridized to the Arabidopsis ATH1 Genome Array (Affymetrix). Raw data can be obtained from NASCarrays, experiment number 668.