The small and total RNA fractions were isolated from the individual tissues using the miRVana™ microRNA Isolation Kit (Applied Biosystems/Ambion, Austin, TX, USA) and TRI REAGENT® (Molecular Research Center, Inc., USA), respectively
Label
Hy5
Label protocol
A total of 200 ng and 1μg of the small RNA and total RNA fraction was used for each sample, respectively. 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
The miRCURY™ LNA microRNA Microarray version 9.2 was used for the array studies. Hybridization was performed in the hybridization station (Tecan Group Ltd., Männedorf, Switzerland), for 16 hours followed by stringent 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
F50_cortex_Hy5_Total RNA
Data processing
The text files, generated by Imagene v.7.0, were imported into the R environment (R Development Core Team (2007). The importing and pre-processing of data was performed using the Linear Models for Microarray Data (LIMMA) package [25]. The “normexp” background correction method [25] was applied. The intensities were then LOG 2 transformed and normalized, using the LIMMA implementation in quantile normalization. The intensities of four intra slide replicates were used to calculate average intensities of each hybridization signal. The data were filtered, firstly to include only human and porcine microRNA (hsa and ssc microRNAs, respectively) and secondly to exclude the probes which: a) showed little or no variation; b) their intensities in all cases were close to the background c) had more than 5 not available values; d) had range of linear intensities exceeding 100. The final, filtered data set consisted of intensity values for 240 probes (see supplementary data for complete list).
