All patients had archival formalin-fixed, paraffin-embedded, (FFPE) tumor tissue stored in the dark
Extracted molecule
total RNA
Extraction protocol
Five sections of 10 μm were de-paraffinized and total RNA was extracted with Roche high pure kit according to the manufacturer’s protocol (Roche, Basel, Switzerland).
Label
Hy3
Label protocol
A common reference design, where the common reference contained an equimolar mixture of total RNA from all samples, per set, was applied to enable both one and two-channel analysis of the data. The sample channel was labeled with Hy3™ dye, while the reference channel was labeled with Hy5™ dye according to the manufacturer’s recommendation (Exiqon A/S, Vedbaek, Denmark).
Hybridization protocol
Labeled RNA was hybridized overnight to miRCURY LNA™ microRNA arrays discovery version 9.2, 11.0 and 11.5, for the 3 sets, respectively, on a Tecan HS Pro 4800 hybridization station
Scan protocol
After hybridization, the rinsed and dried arrays were scanned in a DNA microarray scanner (Agilent, model G2565BA, California, USA).
Data processing
The data was both treated as one channel data using the Hy3 signal intensities, and as two-channel data using the Hy3/Hy5 ratios. Here, we only present the one-channel data, as the ratio analysis gave comparable results. Initially, background subtraction was performed in R v.2.2.1 using the backgroundCorrectmethod in the LIMMA (Linear Models for Microarary Data) package, which is available as part of the Bioconductor project (http://www.bioconductor.org). The intensity values were then log2-scaled and the median probe signals were quantile normalized. Thus, the statistical analysis is based on the quantile normalized log2-scaled Hy3 signals.
