Total RNA was isolated from the individual cell lines using Trizol (Invitrogen)
Label
Hy3
Label protocol
A total of 1000 ng of total RNA was used for each sample. MiRCURY™ LNA microRNA Power labeling Kit (Exiqon, Vedbaek, Denmark) was used following the manufacturers’ recommendations. Spike-ins (used as control probes) were added in equal amounts to each reaction and labeled.
Hybridization protocol
Hybridization was performed in the hybridization station (Tecan Group Ltd., Männedorf, Switzerland), for 16 hours followed by stringency washes.
Scan protocol
Subsequently, slides were dried and scanned. The analysis were performed using the relevant GenePix® Array Lists (GAL files) www.exiqon.com. The arrays were scanned by the Agilent scanner (Agilent Technologies, Santa Clara, CA, USA), to generate Tagged Image File Format (TIFF) images. 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.
Description
KM_12_COL
Data processing
The text files, generated by Imagene v.8.0, were imported into Rosetta Resolver and normalized as previously described. The data used are median of replicate spots from normalized values using the Quantile normalization method in limma package in R version 11 as descriped in Søkilde et al. (MICRORNA EXPRESSION ANALYSIS BY LNA ENHANCED MICROARRAYS)
