Nucleospin 2 kit

Total RNA was isolated from the spleens from 52-week-old mice using NucleoSpin II kit (Macherey-Nagel) according to the manufacturer’s instructions, and 50 ng was used to determine the absolute levels of gene expression. Hybridization and nCounter were performed according to the manufacturer’s protocol (Nanostring Technologies, Seattle, WA, USA). In brief, reactions were hybridized for 20h at 65°, after which the products were used to run on the nCounter preparation station for removal of excess probes. Data were collected with the nCounter digital analyzer by counting individual barcodes. Data generated from the nCounter digital analyzer were examined with the nCounter digital analyzer software system v2.1.1 (Nanostring Technologies). Data were normalized to the geometric means of spiked-in positive controls (controls for assay efficiency) and spiked-in negative controls (normalized for background). The data were further normalized to the housekeeping genes Gapdh, Hprt, and Tubb5 and are reported as normalized RNA counts (means ± SEM). Nanostring RNA counts were analyzed with the Partek Genomic Suite (Partek, Inc., St. Louis, MO, USA), to identify significantly regulated probe. Heatmaps of Nanostring data were generated with the Partek Genomic Suite.

At the end of the incubations described above, total RNA was extracted (NucleoSpin II Kit; Macherey-Nagel, Bethlehem, PA, USA). For PCR, reverse transcription was performed using RT² First Strand Kit and quantitative PCR was performed (iCycler iQ; BioRad, Hercules, CA, USA), using primers relevant to psoriasis and/or retinoic acid signalling. Gene expression was standardized to the housekeeping gene peptidylprolyl isomerase A (PPIA). All PCR reagents were from Qiagen (Germantown, MD, USA).
For the DNA microarray experiment, gene expression was profiled using an array platform (Human Genome U133 Plus 2.0; Affymetrix, Santa Clara, MD, USA), containing > 470 000 transcripts associated with 20 150 human genes. False discovery rate (FDR), was controlled for by adjusting raw P values derived from linear models using the Benjamini–Hochberg method.¹⁴ Enrichment of differentially expressed genes with respect to ordered gene lists was performed using the Wilcoxon rank sum test.^{15 (link)}