Genechip whole transcript sense target labeling assay manual

Global gene expression analyses of aorta specimens from WT and eNOS^−/− mice were performed using Affymetrix GeneChip^Ⓡ Mouse Gene 2.0 ST Array (Affymetrix, Santa Clara, CA, USA). Sample preparation was performed according to the instructions and recommendations provided by the manufacturer. In brief, 300 ng of total RNA from each sample was converted to double-strand cDNA using a random hexamer incorporating a T7 promoter. Amplified RNA was generated from the double-stranded cDNA template through an in vitro transcription reaction and purified with the Affymetrix sample cleanup module. cDNA was regenerated through random-primed reverse transcription using a dNTP mix which contained dUTP. The cDNA was then fragmented by UDG and APE 1 restriction endonucleases, and end-labeled by terminal transferase reaction incorporating a biotinylated dideoxynucleotide. Fragmented end-labeled cDNA was hybridized to the GeneChip^Ⓡ Mouse Gene 2.0 ST arrays for 17 hr at 45°C as described in the Gene Chip Whole Transcript Sense Target Labeling Assay Manual (Affymetrix). After hybridization, the chips were stained, washed in a Genechip Fluidics Station 450 (Affymetrix) and scanned using a Genechip Array scanner 3000 7G (Affymetrix) (25 (link)).

For gene expression analysis, the mice were sacrificed at the age of 12–20 weeks, and their thoracic aorta were snap frozen and pestle ground under liquid nitrogen. Trizol reagent (Invitrogen) was used for the extraction of RNA from the powdered tissue. The gene expression profiling of the tissues from homozygous mgR and WT mice was performed using Affymetrix Genechip Mouse Gene 1.0 ST microarray (Affymetrix, Santa Clara, CA). Briefly, biotinylated cDNAs were synthesized from equal amounts of total RNA. After cDNA probes were hybridized as described in the Gene Chip whole transcript sense target labeling assay manual (Affymetrix), the chips were scanned using a Genechip Array scanner 3000 7G (Affymetrix) and the scanned images were analyzed using the Affymetrix Command Console software (version1.1). Probes with p value of <0.05 were used for analysis and were normalized using the Robust Multi-array Average normalization method.

Global gene expression analyses using Affymetrix GeneChip^® Human Gene 2.0 ST oligonucleotide arrays were performed by the commercial microarray service Ebiogen (Korea). Total RNAs from shCTL HeLa cells and shUHRF1 HeLa cells (300 ng from each sample) was converted to double-stranded cDNA using random hexamers incorporating a T7 promoter and Fragmented cDNA was generated by manufacturer’s protocol (Affymetrix, USA). Fragmented end-labelled cDNA was hybridised to the array for 16 h at 45°C and 60 rpm, as described in the GeneChip Whole Transcript Sense Target Labeling Assay Manual (Affymetrix). The chip was scanned with a GeneChip Array Scanner 3000 7G (Affymetrix) and analysed using Affymetrix Command Console software, v1.1. Normalisation was performed with the Robust Multi-array Average (RMA) algorithm, implemented in Affymetrix Expression Console software, and graphs and heatmaps were prepared using the MeV program.