Agilent dna microarray scanner g2565ca

Total RNA was extracted from epicotyls of young seedlings grown for 7 d under 30°C/25°C (positive DIF) and 25°C/30°C (negative DIF) (DT/NT, 16 h photoperiod) as described in the preceding subsection. Three biological replicates were used for microarray analysis. Target labeling was performed according to the manual of the Low Input Quick Amp Labeling Kit, One-Color (Agilent Technologies). We used a tomato custom-designed microarray (platform ID “GPL21511”). Hybridization was performed according to the manufacturer’s instructions. We scanned the microarray images using an Agilent DNA Microarray Scanner G2565CA (Agilent Technologies). Scanned images were converted to signal data using Feature Extraction software (Agilent Technologies). Value definition in the data matrix was Log2. GO enrichment analysis among DEGs (top 300 upregulated and top 300 downregulated genes; P < 0.05) was performed using the GO Analysis Toolkit and Database for Agricultural Community (AgriGO, http://systemsbiology.cau.edu.cn/agriGOv2/) (Tian et al., 2017 (link)).

To investigate DNA copy number changes on bulk tumors, we used the Mouse 244 k Custom Oligo platform (GPL15359 Agilent UNC Perou Lab 1 × 244 k Custom Tiling CGH Array)^{69 (link)}. Labeling and hybridization were performed according to the manufacturer’s instructions using the Agilent Genomic DNA Labeling Kit PLUS (Catalog Number 5188–5309). One microgram of DNA from liver or spleen of FVB strain mouse was used as normal reference DNA, which was compared versus 1 μg of DNA from C3Tag DCIS and tumor samples. Microarrays were scanned on an Agilent DNA Microarray scanner (G2565CA) and the data uploaded to the University of North Carolina Microarray Database (www.genome.unc.edu). To determine regions of Copy Number Aberration (CNA), we utilized the R package SWITCHdna^{18 (link),70 (link)}.

Per sample, 100 ng of total RNA was combined with Array Control RNA spikes (Ambion) and labeled using a low input Quick Amp whole-transcript (WT) labeling kit (Agilent) according to the manufacturer’s instructions. Each hybridization mixture comprised 1.1 μg test (Cy3) and 1.1 μg reference (Cy5) components, representing a common reference pool of all samples. The samples were dried, and 1.98 μl water was added. The hybridization cocktail was made according to the manufacturer’s instructions (NimbleGen Arrays User’s Guide—Gene Expression Arrays Version 5.0, Roche NimbleGen). Then, 7.2 μl from this mix was added to each sample. The samples were incubated for 5 min at 65°C and 5 min at 42°C prior to loading. Hybridization samples were loaded onto a 12-by-135,000 custom microarray designed against Bacillus subtilis (Roche NimbleGen, Inc.) as used in previous studies (4 (link), 46 (link)). Microarrays were hybridized for 20 h at 42°C with a NimbleGen hybridization system (Roche NimbleGen, Inc.). Afterward, the slides were washed according to the NimbleGen Arrays User’s Guide—Gene Expression Arrays Version 6.0 and scanned with an Agilent G2565CA DNA microarray scanner (Agilent Technologies).