Microarray Analysis of LEP-Treated Samples

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.