tissue: rectal tumor t (tumor) or n (normal): T patient identifier: P100
Extracted molecule
total RNA
Extraction protocol
RNA was extracted using TRIZOL (Invitrogen, Carlsbad, CA). Nucleic acid quantity, quality and purity were determined using a spectrophotometer (Nanodrop, Rockland, DE) and a 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA).
Label
Hy3
Label protocol
One µg of total RNA from each sample was labeled using the miRCURY™ LNA microRNA Power labeling kit (Exiqon) following a two-step protocol: First, Calf Intestinal Alkaline Phosphatase (CIAP) was applied to remove terminal 5'phosphates; second, fluorescent labels were attached enzymatically to the 3'-end of the microRNAs. Sample-specific RNA was labeled with Hy3 (green channel) fluorophore.
Hybridization protocol
Hybridization was performed in the hybridization station (Tecan Group Ltd., Männedorf, Switzerland) for 16 hours, followed by stringency washes. Subsequently, slides were dried and scanned.
Scan protocol
The arrays were scanned by the Agilent scanner (Agilent Technologies, Santa Clara, CA, USA) to generate Tagged Image File Format (TIFF) images. The analysis was performed using the relevant GenePix® Array Lists (GAL files) (www.exiqon.com). Image analysis: The intensities were converted to digital values using Imagene version 7.0 software. The quality control of the spots was performed by the software and adjusted manually.
Data processing
Data processing and normalization: Low-level analysis was performed in the R statistical computing environment, including importing and pre-processing of the data using the LIMMA package (available at http://bioconductor.org). After excluding flagged spots from the analysis, the “normexp” background correction method, plus offset=50 was applied. The intensities were log2 transformed and quantile normalized as implemented in LIMMA. Log2 intensities (single channel analysis) of four intra-slide replicates were averaged.
