Agilent sureprint g3 mouse ge 8x60k microarray

RNA from the liver of newborn mice was purified according to standard methods (SI M & M). Samples were amplified, labeled using the Agilent Quick Amp labeling kit, and hybridized using the Agilent SurePrint G3 Mouse GE 8x60K Microarray (G4852A-028005) (Agilent Technologies, Palo Alto, CA) and Agilent SureHyb hybridization chambers. Three independent replicate experiments were performed for each group. The resulting working gene lists of transcripts were imported to the Ingenuity® iReport microarray analysis program (Ingenuity® Systems) and FatiGo for gene ontology [19 (link)].

Microarray experiment procedures were carried out following the manufacturer’s protocols. Total RNA was amplified by an Agilent Quick Amp Labeling Kit (Agilent Technologies, USA). Cy-labeled cRNA (0.3 μg) was cleaved to an average size of about 50–100 nucleotides by incubation with fragmentation buffer (Agilent Technologies) at 60 °C for 30 min. Equal Cy-labeled cRNA was pooled and hybridized to Agilent SurePrint G3 Mouse GE 8x60K Microarray (Agilent Technologies, USA), then scanned by an Agilent microarray scanner (Agilent) at 535 nm for Cy3 and 625 nm for Cy5. Scanned images were analyzed by Feature Extraction software 10.5 (Agilent Technologies), and image analysis and normalization software was used to quantify signal and background intensity for each feature, which substantially normalized the data using the rank-consistency-filtering LOWESS method.

Total RNA (0.2 μg) was amplified by a Low Input Quick-Amp Labeling kit (Agilent Technologies) and labeled with Cy3 (CyDye, Agilent Technologies) during the in vitro transcription process. Cy3-labeled complementary (c)RNA (0.6 μg) was fragmented to an average size of about 50~100 nucleotides by incubation with fragmentation buffer at 60°C for 30 min. Corresponding fragmented, labeled cRNA was then pooled and hybridized to an Agilent SurePrint G3 Mouse GE 8 x 60K Microarray (Agilent Technologies) at 65°C for 17 h. After washing and drying by blowing with a nitrogen gun, microarrays were scanned with an Agilent microarray scanner (Agilent Technologies) at 535 nm for Cy3. Scanned images were analyzed, and normalization software was used to quantify the signal and background intensities of each feature. Microarray dataset of Tg-3m and Tg-6m tumors was deposited in public database Dryad (http://datadryad.org/; doi:10.5061/dryad.n01tb). Knowledge-based Gene Set Enrichment Analysis (GSEA) software was used to analyze the experimental datasets.