Total RNA was isolated from tissues with the mirVana RNA Isolation labeling kit (Ambion, Inc.) according to the manufacturer's protocol. RNA quality and integrity were confirmed by the ExperionTM Automated Electrophoresis System (BIO-RAD).
Label
Biotin
Label protocol
Biotin-labeled cRNA samples for hybridization were prepared according to Illumina's recommended sample labeling procedure: 500 ng of total RNA was used for cDNA synthesis, followed by an amplification/labeling step (in vitro transcription) to synthesize biotin-labeled cRNA using the Illumina TotalPrep RNA Amplification kit (Ambion Inc., Austin, TX). cRNA concentrations were determined using a spectrophotometer (Nanodrop, ND-1000).
Hybridization protocol
Labeled, amplified material (750 ng per array) was hybridized to the Illumina HumanHT-12 V4.0 expression BeadChip according to the manufacturer's instructions (Illumina, Inc., San Diego, CA).
Scan protocol
Arrays were scanned with an Illumina BeadArray Reader confocal scanner (BeadStation 500GXDW; Illumina, Inc., San Diego, CA) according to the manufacturer's instructions.
Data processing
Array data processing was performed on Illumina BeadStudio software. We normalized gene expression data using quantile normalization and log2 transformation. To export to a data matrix, the Sample Gene Profile option of this software was used.
