Affymetrix genechip expression analysis technical manual

The FDA Salmonella custom high-density Affymetrix DNA microarray platform was used, as previously described [34 (link),35 (link)]. An 8 μg aliquot of purified genomic DNA was fragmented by incubation at 37 °C for 10 min in a 50 μL reaction containing 1× One-Phor-All Plus Buffer [Tris, Magnesium and Potassium acetate (Ratios 1:1:5)] and 0.1 units DNase I (GE Healthcare, Pittsburg, PA, USA). Following fragmentation, the DNA was labeled at the 3′-end using 1 mM biotin-11-ddATP (PerkinElmer NEL508, Waltham, MA, USA), 5X terminal transferase buffer (Promega, Madison, WI, USA), and 60 units of terminal transferase enzyme (Promega), as described earlier. The genomic DNA samples were hybridized following the Affymetrix GeneChip Expression Analysis Technical Manual (Affymetrix, Santa Clara, CA, USA, 2014), washed in the Affymetrix FS-450 fluidics station (Affymetrix, Santa Clara, CA, USA), and scanned using software of the Affymetrix GeneChip Command Console (AGCC) Scanner 3000. Reagents used in hybridization, washing, and staining were prepared according to the Affymetrix GeneChip Expression Analysis Technical Manual [36 ]. For microarray data analysis, a probe set intensity for each allele represented on the microarray chip were assessed using the Robust MultiArray Averaging (RMA) function in the Affymetrix package of R-Bioconductor [37 (link)].

We previously reported the experimental procedures to perform whole-genome transcript profiling [39 (link)]. Briefly, we isolated total RNA from Caco-2 cells after decitabine (3 µM), 4-PBA (3 mM) and the combined treatment and followed the Affymetrix Gene Chip® Expression Analysis Technical Manual (Affymetrix, USA). We prepared cDNA followed by an in vitro transcription step to obtain copy RNA. Based on metal-induced hydrolysis we obtained fragmented cRNA, which we hybridized onto the Affymetrix Gene chip HG-U133 version 2.0 array (human). After scanning of the arrays, we normalized the signal intensity data with the robust multi-array average (RMA) algorithm of the Gene Expression Console software for background-adjusted and log-transformed perfectly matched individual probes. Subsequently, we uploaded the data onto the GeneXplain 3.0 platform (http://platform.genexplain.com/bioumlweb) and computed t-test for statistical analysis of differentially expressed genes (DEGs) by comparing DMSO vehicle controls against the various treatments. Only DEGs adjusted for the false discovery rate (FDR) p < 0.05, and fold change (FC) > 1.5 was considered for further analysis. Essentially, we performed the miRNA gene expression analysis as described above and more detailed in our previous publication [39 (link)].

At the end of high temperature stress, fresh flag leaves were used for gene transcriptome analysis with the Affymetrix GeneChips (Affymetrix, Santa Clara, CA, USA). The analysis was conducted in the Capitalbio Corporation in Beijing according to the instructions for Affymetrix (Affymetrix GeneChip Expression Analysis Technical Manual, Affymetrix). GeneChip Operating Software 1.4 was used to analyze the hybridization data under the criteria of at least 2.0-fold changes (log₂ values), with a false discovery rate <0.05 at gene expression level. Functional annotation was performed based on Arabidopsis thaliana genome databases.