Affymetrix genechip command console software agcc

One hundred and fifty nanogram of each RNA sample were processed using Affymetrix GeneChip Whole Transcript (WT) PLUS Reagent Kit (Affymetrix, Santa Clara, CA, USA). Fifteen microgram of cRNA was input into the second cycle cDNA reaction. 5.5 μg of ss‐cDNA were input for fragmentation. Each DNA fragment was end labeled with biotin using terminal deoxynucleotidyl transferase before being hybridized to the arrays. Hybridization cocktails were prepared and applied to Gene Chip Human Gene 1.0 ST arrays (Affymetrix, Santa Clara, CA). Hybridization was performed at 60 rpm for 16 h at 45°C. Wash and stain processes were performed with the Genechip Expression Wash and Stain Kit in the GeneChip Fluidics Station 450 (Affymetrix, Santa Clara, CA). The probe arrays were scanned using the GeneChip Scanner 3000 7G and Affymetrix GeneChip Command Console Software (AGCC) (Affymetrix, Santa Clara CA, USA) to produce.CEL intensity files.

All procedures were performed in accordance with the guidelines in the Affymetrix Expression Analysis Technical Manual (Affymetrix Inc., Santa Clara, CA, USA). Total RNAs were labeled using the FlashTag Biotin HSR RNA Labeling Kit (Thermo Fisher). The GeneChip^® Affymetrix miRNA microarray (Affymetrix Inc.) was used to hybridize the biotin-labeled samples. The Affymetrix GeneChip^® scanner was used to examine all arrays, and raw data analysis was carried out using the Affymetrix GeneChip^® Command Console^® Software (AGCC) (Affymetrix Inc., Santa Clara, California, USA). The measured intensity of each array probe was obtained from CEL files, which contained the raw data images developed by the scanner. A p-value of <0.05 and fold change of >2 served as cut-off criteria to restrict a broad range of differentially expressed microRNAs.