Agilent high resolution c scanner

Genome-wide miRNA expression microarray experiments (n = 5 for 2-, 5-, 6-, 15-, 21-, and 78-week males and 6-, 15-, 78-, and 104-week females; n = 4 for 8- and 104-week males and 2-, 5-, 8-, and 21-week females) were completed for a total of 74 microarrays. Single color (Cy3) Agilent Rat 8 × 15 K miRNA microarrays and reagents were used according to the manufacturer’s protocols (Agilent Technologies, Santa Clara, CA, USA) using 100 ng of total RNA. An Agilent one-color spike-in kit was used as a positive control to measure both labeling and hybridization efficiency. Labeled probe was purified using BioRad Micro Bio-Spin 6 columns (BioRad Life Science Research, Hercules, CA, USA), according to the manufacturer’s protocol. Hybridized microarrays were scanned using the Agilent High Resolution C Scanner (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s protocol. The images were analyzed using Agilent’s Feature Extraction software. Expression data were deposited in Gene Expression Omnibus (Accession ID GSE64842).

To investigate rearrangements in higher resolution, we analyzed DNA from patients 2, 3, 4 and 5 with a custom designed CytoSure DMD array, 4 × 44K (Oxford Gene Technology, IP, UK). This array consisted of 44,000 60-mer oligonucleotides, with an average probe spacing of 10 bp within the exons and 106 bp within introns. The reactions were performed according to the manufacturer's protocol using 200 ng of purified genomic DNA. The slides were scanned on an Agilent High-Resolution C Scanner (Agilent, Santa Clara, CA) and analyzed using CytoSure Interpret software (Oxford Gene Technology IP, UK).
To determine the parent of origin of the de novo noncontiguous duplication in patient 3, we genotyped the patient and parents with the Illumina Human 660W-Quad BeadChip.

Microarray analysis was performed in samples obtained from Ad5-porIFNα- and PBS-treated animals at 1 day post treatment, prior to challenge with EBOV. Total RNA was extracted from PBMC and BALP, and the concentrations were determined using a NanoDrop (NanoDrop Technologies) and shipped to MOGENE LC (St. Louis, MO, USA) for the microarray assay. Microarray analysis was performed using an Agilent porcine 4x44K microarray (Agilent Technologies) and following the manufacturer’s protocols. Microarray slides were scanned on an Agilent high resolution C scanner and images processed using the Agilent Feature Extraction software. The quality of samples was analyzed using the ArrayQualityMetrics package and the data imported into GeneSpring 14.8 (Agilent) for further analysis. The data was normalized using a shift to the 75th percentile; thereafter, the value was further transformed as log2. All gene IDs from genes that were up- or down-regulated > 2-fold were then imported into the DAVID bioinformatics suite (https://david.ncifcrf.gov/, accessed on 26 July 2019) for detecting overrepresentation of signaling pathways from the Kyoto Encyclopedia of Genes and Genomes (KEGG) [33 (link)] and gene ontologies (GO) [34 (link)].
FDR-values and p-values were calculated by the DAVID bioinformatics suite using an adapted method for multiple testing with independent statistics [35 (link)].