Affymetrix genechip scanner 3000 7g

We analyzed the gDNA with the Affymetrix GeneChip® Genome-Wide Human SNP Array 6.0 (Santa Clara, CA, USA). This array contains 906,600 SNP probes and 946,000 nonpolymorphic oligonucleotides; the median intermarker distance over all 1.8 million SNP and copy number markers combined is less than 700 bases.
The procedures for DNA digestion, ligation, PCR amplification, fragmentation, labeling, denaturing and hybridization into the array were performed in 147 DNA samples (two DNA samples were not included because they did not pass quality controls) according to the protocols provided by the supplier. Arrays were stained and washed in the Affymetrix GeneChip Fluidic Station 450 and scanned using an Affymetrix GeneChip Scanner 3000 7G (Affymetrix, Santa Clara, CA, USA). We analyzed the files obtained with the appropriate bioinformatics tools.

Differential gene expression of P. aeruginosa biofilm cells caused by 6-gingerol was analyzed using GeneChip P. aeruginosa Genome Array (900339, Affymetrix, CA, USA). A PAO1 chip has been used to analyze both PAO1 and PA14 transcriptome because of the high similarity of genomes between the two strains46 (link). All of the procedures followed the manufacturer's protocols (Affymetrix) and were conducted at Seoulin Bioscience (Seongnam, South Korea). Briefly, total RNA (10 μg) was converted into cDNA using random primers, and the cDNA was fragmented using DNase I and biotinylated using terminal transferase. Biotinylated cDNA (5 μg) was then hybridized for 16 hr at 45°C on the GeneChip. An Affymetrix Fluidics Station 450 (Affymetrix) was used to wash and stain the GeneChip, and Affymetrix GeneChip Scanner 3000 7G (Affymetrix) was used to read the hybridization signals of the GeneChip. The Robust Multi-chip Average (RMA) algorithm was used for normalizing probe-level intensity47 (link).
Microarray Suite version 5.0 (Affymetrix) was used to analyze GeneChip data using default analysis settings and global scaling. The differentially expressed genes were selected based on greater than 1.5-fold repression or induction by 6-gingerol.

Total RNA was used to synthesize double-stranded complementary DNA (cDNA) utilizing a random priming method, and then double-stranded cDNA was fragmented, labelled and hybridized to the Affymetrix GG-H Array. After hybridization and washing, processed slides were scanned with the Affymetrix GeneChip Scanner 3000 7G (Affymetrix, Santa Clara, CA). The Affymetrix Expression Console (version 1.2.1) implementation of RMA was used for quantile normalization and background correction. All gene level files were imported into Affymetrix Expression Console (version 1.2.1) and normalized by the quantile method. Combat Software was used to adjust the normalized intensity to remove batch effects. A random variance model (RVM) [23] (link)
t-test was applied to discriminate differentially expressed genes between controls and MDD patients because the RVM t-test can raise degrees of freedom effectively in the case of small samples. After false-discovery rate (FDR) analysis, we selected differentially expressed genes only if p value <0.05 and the change in expression was ≥1.5-fold. Hierarchical clustering was performed using EPCLUST. The microarray analysis was performed by Gminix, Shanghai, PR China.

Total RNA was isolated from each hepatic, WAT, and BAT sample by TRIzol reagent (Invitrogen Japan, Tokyo, Japan) and purified using an RNeasy mini kit (Qiagen, Tokyo, Japan). The RNA Integrity Number (RIN) was estimated as an index of the quality of the total RNA using an Agilent 2100 Bioanalyzer (Agilent Technologies Japan, Tokyo, Japan). The values for RIN were over 8.3.
Then, amplified RNA (aRNA) was synthesized from 200 ng of purified total RNA, and biotinylated aRNA was obtained using a GeneChip® 3′IVT Express Kit (Affymetrix, Santa Clara, CA, USA). The aRNA was fragmented and hybridized to a GeneChip® Mouse Genome 430 2.0 Array (Affymetrix) for 16 h at 45 °C. The arrays were washed and stained with phycoerythrin using the GeneChip® Fluidics Station 450 (Affymetrix). The arrays were submitted to scanning on an Affymetrix GeneChip® Scanner 3000 7G (Affymetrix). The Affymetrix® GeneChip® Command Console® Software (Affymetrix) was used to make CEL files.

We performed the analysis of the gDNA with Affymetrix CytoScan 750K Arrays (Affymetrix, Santa Clara, CA, USA) on the three patients. This array provides genome-wide coverage, including 550,000 markers for detecting copy number variation and 200,436 SNP probes. We performed the procedures for DNA digestion, ligation, PCR amplification, fragmentation, labelling, denaturing and hybridization into the array according to the protocols and QC guidelines provided by the supplier. Arrays were then stained and washed in the affymetrix GeneChip Fluidics Station 450 and scanned using an Affymetrix GeneChip Scanner 3000 7G (Affymetrix, Santa Clara, CA, USA); we analyzed the files obtained with the appropriate bioinformatics tools.