breed: crossbreed between Landrace, Yorkshire and Duroc tissue: cerebellum developmental stage: gestation day 100
Extracted molecule
total RNA
Extraction protocol
The small RNA fractions were isolated from the individual tissues using the miRVana™ microRNA Isolation Kit (Applied Biosystems/Ambion, Austin, TX, USA).
Label
Hy3
Label protocol
A total of 200 ng of small RNA fraction 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
The miRCURY™ LNA microRNA Microarray version 11.0 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
Slides were dried and scanned. The analysis was 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
F100_cerebellum_small RNA fraction
Data processing
The text files, generated by Imagene v.8.0, were imported into Rosetta Resolver and normalized as previously described: Book: MicroRNA Profiling in Cancer: A Bioinformatics perspective. 2009. Editor Yuriy Gusev. Chapter: Søkilde R, Kaczkowski B, Barken K B, Mouritzen P, Møller S, Litman T. "MicroRNA Expression Analysis by LNA Enhanced Microarrays". Data was filtered by using probes showing a standard deviation (SD) above 0.1. The final, filtered data set consisted of intensity values for 1088 probes. All clustering and statistical analysis was performed in TMeV. PCA plot of samples was performed using all 1088 probes, by using a median centering of the data set. For the two-way hierarchical clustering, the 181 probes on the array annotated as pig microRNAs was used (see the 'GSE24106_pig_annotation.txt' supplementary file). In case of lack of annotation in pig, human probes were used. The 1- Pearson correlation coefficient was used as a distance metric. Each probe is mean-centered and compared to the mean expression.
