Computational protocol: Modulation of dendritic cell alternative activation and function by the vitamin A metabolite retinoic acid

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Protocol publication

[…] RNA was extracted from cells using TRIzol or Pure Link RNA Mini Kit and DNase-treated according to the manufacturer’s instructions (Life Technologies, UK). RNA was converted to cDNA using Superscript-III (Invitrogen, UK). Quantitative PCR was performed using SYBR Green mastermix-II (Roche, UK) and a Roche Light Cycler 480, using primers previously documented () with the exception of Aldh1a2F:5′-CATGGTATCCTCCGCAATG-3′ and Aldh1a2R:5′GCGCATTTAAGGCATTGTAAC-3′. To assess DC gene expression, RNA was labelled using the TotalPrep RNA Amplification kit (Life Technologies) and hybridized to Illumina MouseWG-6 BeadChip arrays (MouseWG6_V2_0_R3_11278593_A, each comprising 45281 features), according to the manufacturer’s instructions. Three biological replicates were carried out for wild-type (WT) DCs. All analyses were conducted in R using Bioconductor packages (www.r-project.org; www.bioconductor.org). A total of six arrays were quality controlled (QC) analysed using the arrayQualityMetrics package in Bioconductor (, ). Raw data from arrays that passed QC were transformed using a variance-stabilizing transformation method prior to normalization across all arrays using the robust spline normalization method, using the lumi package in Bioconductor. Pairwise group comparisons were undertaken using linear modelling. Subsequently, empirical Bayesian analysis was applied, including vertical (within a given comparison) P value adjustment for multiple testing, which controls for false discovery rate, using the limma Bioconductor package (). The data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus () and are accessible through GEO Series accession number GSE59868 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE59868). […]

Pipeline specifications

Software tools arrayQualityMetrics, lumi, limma
Databases GEO
Application Gene expression microarray analysis
Organisms Mus musculus
Chemicals Acetaldehyde, Tretinoin, Vitamin A