Affymetrix geneatlas fluidics station

The 100 ng of mRNA from each pooled sample was transferred to succeeding microarray procedure described in details in our recent paper [75 (link)]. Briefly, primary cDNA was obtained and amplified (Ambion^® WT Expression Kit, Ambion Inc., Thermo Fisher Scientific, Waltham, MA, USA). Then, cDNA was labelled with biotin and fragmented (Affymetrix GeneChip^® WT Terminal Labeling and Hybridization, Affymetrix, Santa Clara, CA, USA). Resulting fragments were hybridized to the microarray strips (Affymetrix^® Porcine Gene 1.1 ST Array Strip, Affymetrix), which were afterwards washed, stained according to the manufacturer’s protocol (Affymetrix GeneAtlas Fluidics Station, Affymetrix), scanned (Imaging Station of the GeneAtlas System, Affymetrix) and analyzed (Affymetrix GeneAtlasTM Operating Software, Affymetrix), resulting in generation of raw CEL files containing all of the data.
Downstream analyses were performed, and graphs were obtained using Bioconductor and R programming languages, as well as the Robust Multiarray Averaging (RMA) algorithm. Statistical significance of analyzed genes was determined with the use of moderated t-statistics from the empirical Bayes method, with obtained p-value corrected for multiple comparisons using Benjamini and Hochberg’s false discovery rate. Genes regarded as differentially expressed presented p-value beneath 0.05 and expression higher than 2-fold.

The microarray study was performed as described in detail elsewhere (15 (link)–18 (link)). The total RNA isolated previously was pooled into four samples per group (control, ACTH, forskolin and adropin) treated as described above (see Cell culture and Treatment section). The protocol including in vitro transcription, biotin labeling, and cDNA fragmentation was performed using the Affymetrix GeneChip IVT Express Kit (Affymetrix, Santa Clara, CA, USA). Then the biotin labeled cDNA were hybridized with the Affymetrix Gene Chip Human Genome U219 microarrays together with appropriate internal controls. The hybridization was performed in the AccuBlockTM Digital Dry Bath hybridization oven (Labnet International, Inc., Edison, NJ, USA) at 45°C for 16 h. Subsequently, the microarrays were washed and stained by means of the Affymetrix GeneAtlas Fluidics Station (Affymetrix, Santa Clara, CA, USA). The microarrays were scanned using the Imaging Station of the GeneAtlas System. Initial analysis of the scanned microarrays was carried out with Affymetrix GeneAtlas TM Operating Software.

The microarray study was carried out according to the previously described procedure [69 (link),70 (link),71 (link),72 (link)]. The previously isolated RNA was pooled into four samples, representing control group (n = 2) and experimental group (n = 2) where the RNA was isolated after 24 hours from LEP administration (concentration 1 × 10⁻⁶ M). The protocol involving transcription in vitro, biotin labelling and cDNA fragmentation for further hybridization was carried out using the Affymetrix GeneChip IVT Express Kit (Affymetrix, Santa Clara, CA, USA). Obtained biotin-labelled fragments were hybridized with Affymterix GeneChip Human Genome U219 microarrays together with control cDNA and oligo B2. The hybridization process was conducted with the use of the AccuBlockTM Digital Dry Bath (Labnet International, Inc., Edison, NJ, USA) hybridization oven at 45 °C for 16 h. Then the microarrays were washed and stained according to the technical protocol using the Affymetrix GeneAtlas Fluidics Station (Affymetrix, Santa Clara, CA, USA). The array strips were scanned by the Imaging Station of GeneAtlas System. Preliminary analysis of the scanned chips was performed using Affymetrix GeneAtlasTM Operating Software. The quality of gene expression data was verified using the quality control criteria established by the software. Obtained CEL files were imported into downstream data analysis.