3d gene mouse mirna oligo chip

Total RNA were extracted from cancer cell lines using the mirVana miRNA Isolation Kit (Applied Biosystems, Foster City, CA, USA). Extracted total RNA was checked with a Bioanalyzer (Agilent Technologies, CA, USA) and labeled with a 3D‐Gene miRNA labeling kit (Toray Industries, Kamakura, Japan). Labeled RNA were hybridized onto 3D‐Gene Mouse miRNA Oligo chips (Toray, Kamakura, Japan). The annotation and oligonucleotide sequences of the probes conformed to the miRBase Release 19 database (www.mirbase.org). After stringent washes, the fluorescent signals were scanned with a 3D‐Gene Scanner (Toray Industries) and analyzed using the 3D‐Gene Extraction software (Toray Industries). The raw data for each spot was normalized by subtraction with the mean intensity of the background signal determined from the signal intensities of all blank spots with 95% confidence intervals. Measurements for spots with signal intensities greater than two standard deviations (SD) of the background signal intensity were considered to be valid. The relative expression level of a given miRNA was calculated by comparing the signal intensities of the valid spots throughout the microarray experiments. The normalized data were globally normalized per array, so that the median of the signal intensity was adjusted to 25. All data were submitted to the GEO database, under the accession number GSE75482.

Comprehensive miRNA expression analysis was performed with 250 ng of total RNA from tissue samples and 40 ng of total RNA from serum samples using a 3D-Gene miRNA labelling kit and a 3D-Gene Mouse miRNA Oligo Chip (Toray Industries, Tokyo, Japan), which are designed to detect 1265 miRNA sequences registered in miRBase release 19 (http://www.mirbase.org/). A miRNA was considered to be present if the corresponding microarray signal was higher than (mean +2 × SD] the signal of the negative controls, of which the 5% of the top and bottom ranked were eliminated by signal intensity. Once a miRNA was considered present, the mean signal of the negative controls, of which the 5% of the top and bottom ranked were eliminated by signal intensity, was subtracted from the miRNA signal. To normalise the signals among microarrays tested, a global median normalisation method was applied.