Biopsies were frozen in liquid nitrogen until nucleic acid extractionThe population under study included 89 patients prospectively enrolled into the National Cancer Institute of Mexico. All patients included signed informed consent; the protocol was approved by institutional ethics and scientific committees. Immediately after surgical excision, tumor biopsies were analyzed for pathological confirmation and frozen in liquid nitrogen until nucleic acid extraction.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using the MagNAPure Compact Instrument following manufacturer recommendations (Roche Diagnostics GmbH Roche Applied Science Mannheim, Germany). RNA quality was determined by by means of 18S:28S ratio. Hybridization targets were prepared from 250 ng of total RNA and amplified with whole transcriptome amplification kit 2 (Sigma Aldrich, St Louis, MO)
Label
Cy3
Label protocol
Labeling was performed by NimbleGen Systems One Color Llabeling kit (Nimblegen Roche, Mannheim, Germany), following their standard operating protocol. See www.nimblegen.com. (Nimblegen Roche, Mannheim, Germany).
Hybridization protocol
Hybridization was performed by NimbleGen Systems (Nimblegen Roche, Mannheim, Germany), following their standard operating protocol. See www.nimblegen.com. After standard washes, arrays were scanned.
Scan protocol
Scanning was performed byNimblegen MS200 microarray scanner. Nimblegen Roche, Mannheim, Germany, following their standard operating protocol. Scanned images were gridded by using the NimbleScan v2.6 Software (Nimblegen Roche, Mannheim, Germany).
Description
SAMPLE 89
Data processing
The raw data (.pair file) was subjected to RMA (Robust Multi-Array Analysis; Irizarry et al. Biostatistics 4(2):249), quantile normalization (Bolstad et al. Bioinformatics 19(2):185), and background correction as implemented in the NimbleScan software package, version 2.4.27 (Roche NimbleGen, Inc.).
