Human ht 12v4.0 beadchip arrays

Publicly available datasets found on the R2: Genomic Analysis and Visualization http://r2.amc.nl platform (accessed on 1 October 2021) were used for the evaluation of RNF5 expression in NB primary tumor and melanoma samples. The NB dataset (GSE49710) contains the expression profiles of 498 NB specimens (211 female and 287 male patients, with a diagnosis age ranging from 0 to 25 years; the 75% of patients received the diagnosis before 3 years of age) measured with the Illumina HiSeq 2000 RNAseq platform [16 (link)] whereas the melanoma dataset (GSE65904) reports the gene expression profiles of 214 melanoma tumor samples analyzed with Illumina Human HT-12V4.0 BeadChip arrays [17 (link)]. RNF5 differential expression and survival analyses were performed with R2 platform tools, using the median value as a cut-off to define high or low gene expression. The differential expression of RNF5 between high-risk and low-risk NB patients was reported as Log₂ fold change (FC) value and the significance was calculated with ANOVA test. Patient overall survival (OS) and event-free survival (EFS) were assessed by Kaplan-Meier curves. The separation significance of the survival curves was assessed with log-rank test adjusted by the Bonferroni method.

In order to analyze the effects of GARP silencing on the gene expression in ASCs, total RNA (500 ng), isolated from three independent biological replicates of NT, LV‐CTRL, LV#18, and LV#19 ASCs 6 days after transduction, was amplified using the Illumina TotalPrep RNA Amplification Kit (Ambion, Austin, Texas), reverse‐transcribed into first‐ and second‐strand cDNA and cRNA labeled with biotin was generated according to the manufacturer's instructions. The cRNA was hybridized overnight to the Human HT‐12 V4.0 BeadChip arrays (Illumina). Beadchips were washed, stained with dye‐labeled streptavidin, and scanned with the Illumina IScan. Raw data were exported from Illumina GenomeStudio and processed in R using negative control probes for background correction and quantile normalization.44 Probes with detection P‐values <.05 in at least two replicates were discharged, and expression values of the remaining probes corresponding to the same gene were aggregated by the median value. Differential expression analysis was carried out using linear models implemented in the limma R package.45 Genes with an FDR‐adjusted P‐value <.05 and absolute fold‐change >0.5 were selected as significantly differentially expressed. Analysis of gene functions and canonical pathways was performed using the Ingenuity Pathway Analysis (IPA) software (Ingenuity Systems Inc., Redwood City, California).

Total RNA was used as template to amplify and label cRNA using the Illumina TotalPrep RNA Amplification Kit (Ambion, Austin, TX) as per manufacturer's instructions. cRNA synthesis and Illumina BeadChip array probing were performed at the microarray facility at David H. Murdock Research Institute, Kannapolis, NC. The labeled probes were then mixed with hybridization reagents and hybridized overnight to the Human HT-12 V4.0 BeadChip arrays (Illumina, San Diego, CA), permitting analysis of over 47,000 transcripts (http://www.illumina.com/products/humanht_12_expression_beadchip_kits_v4.ilmn). Following washing and staining, the BeadChips were scanned with Illumina IScan to measure fluorescence intensity at each probe. Each “probe” is designed for an isoform of a specific gene product and consists of 12–40 identical beads randomly spread out over the surface of the array. The raw data images were imported into Illumina Genome Studio, which generated an average intensity of each probe for each sample.