Genechip array scanner 3000 7g

Processing of colorectal FFPE RNA samples was performed at the Genomics and Bioinformatics Shared Resource, Cancer Center, University of Hawaii. RNA sample integrity was checked on Agilent 2100 Bioanalyzer using RNA Pico chip. Samples were prepared for microarray hybridization as described in the Thermo Fisher Scientific GeneChip Whole Transcript (WT) Expression manual using the WT Pico Reagent Kit. Double-stranded cDNA was generated from 100 ng of total RNA. Subsequently, cRNA was synthesized using the WT cDNA Synthesis and Amplification Kit (Thermo Fisher Scientific). cRNA was purified and reverse transcribed into single-stranded (ss) DNA. Subsequently a combination of uracil DNA glycosylase (UDG) and apurinic/apyrimidinic endonuclease 1 (APE 1) was used to fragment ssDNA, which was afterwards labeled with biotin (WT Terminal Labeling Kit, Thermo Fisher Scientific). In a rotating chamber, 5.5 μg of fragmented and labeled ss-cDNA were hybridized to the Clariom D Human Array for 16 h at 45 °C. After washing and staining on Affymetrix Fluidics Station FS450 using pre-formulated solutions (Hyb, Wash & Stain Kit, Thermo Fisher Scientific), the hybridized arrays were scanned on the Affymetrix GeneChip Array Scanner 3000-7G. The expression intensity data were extracted from the scanned images and stored as CEL files.

Total RNA was extracted from adherent cells and cell-spheres from N87 and SNU484 gastric cancer cell lines using RNeasy Mini kits (Qiagen, Valencia, CA, USA). Microarray procedures were carried out according to the manufacture0072’s protocols. Briefly, 6-μg aliquots of total RNA were used to prepare double-stranded cDNA. cDNAs were amplified by PCR and labelled with biotin using the IVT labelling kit (Affymetrix, Santa Clara, CA, USA). Labelled cRNA was fragmented and hybridized to an Affymetrix GeneChip Human Genome U133 plus 2.0 high-density oligonucleotide arrays (Affymetrix). Microarrays were then washed using a GeneChip Fluidics Station 450 (Affymetrix) and scanned using a GeneChip Array Scanner 3000 7G (Affymetrix). Expression data were generated using Affymetrix Expression Console software version 1.1 using MAS5 algorithm normalization. Expression intensity data in CEL file was normalized using the MAS5 algorithm to reduce noise.

Total RNA was extracted from adherent cells and cell spheres of SNU245 and SNU1196 cells using RNeasy Miniprep kits (Qiagen, Valencia, CA, USA). Microarray analysis was performed according to the kit’s protocol [19 (link)]. Briefly, double-stranded cDNA was prepared using 6 µg aliquots of total RNA, amplified using polymerase chain reaction (PCR), and labeled with biotin using an IVT labeling kit (Affymetrix, Santa Clara, CA, USA). The labeled cDNA was fragmented and hybridized to an Affymetrix GeneChip Human Genome U133 Plus 2.0 high-density oligonucleotide Array (Affymetrix). The microarrays were then washed using a GeneChip Fluidics Station 450 (Affymetrix) and scanned using a GeneChip Array Scanner 3000 7G (Affymetrix). Expression data were generated using Affymetrix Expression Console software version 1.1 using MAS5 algorithm normalization. The expression intensity data in the CEL file were normalized using the MAS5 algorithm to reduce noise.

Total RNA from human ES cells (hESCs), CiMS-iPSCs, and CIMS were isolated using RNeasy Mini Kit columns (Qiagen, Hagen, Germany). RNA samples hybridized to the GeneChip® Human Gene 1.0 ST arrays and the chips were stained and scanned using a GeneChip Array scanner 3000 7G (Affymetrix, CA, USA). The expression intensity data were extracted from the scanned images using Affymetrix Command Console software version 1.1 and analyzed using the Robust Multi-array Average (RMA) algorithm implemented in the Affymetrix Expression Console software (version 1.3.1.).