Affymetrix expression analysis technical manual

All protocols were conducted as described in the standard Affymetrix Expression Analysis Technical Manual (Affymetrix, Inc., Santa Clara, CA, USA). RNA was labeled using the FlashTag™ Biotin HSR RNA Labeling Kit (Thermo Fisher, Waltham, MA, USA). Biotin-labeled samples were hybridized to the GeneChip^® Affymetrix miRNA microarray (Affymetrix). All arrays were scanned using the Affymetrix GeneChip^® scanner, and raw data analysis was performed using the Affymetrix GeneChip^® Command Console^® Software (AGCC). The raw data images generated from the scanner were processed into CEL files containing the measured intensities for each probe on the array. A fold change of >2 and p-value of <0.05 was used as a cut-off to screen a wide range of differentially expressed miRNAs.

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.

Microarray services were provided by the Penn Microarray Facility. All protocols were conducted as described in the standard Affymetrix Expression Analysis Technical Manual (Affymetrix Inc., Santa Clara, CA). Briefly, biotinylated cRNA was prepared from 100 ng total RNA; following fragmentation, cRNA was hybridized for 16 h at 45°C on the Affymetrix miRNA-specific arrays. Microarrays were then washed at low (6X SSPE) and high (100 mM MES, 0.1 M NaCl) stringency and stained with streptavidin-phycoerythrin in an Affymetrix Fluidics Station 400. Fluorescence was amplified by adding biotinylated anti-streptavidin and an additional aliquot of streptavidin-phycoerythrin stain. A confocal scanner (Hewlett-Packard Gene Array Scanner G2500A) was used to collect fluorescence signal after excitation at 570 nm.