Agilent microarray scanner g2565ca

Array-CGH was performed using an Agilent Sureprint G3 CGH+SNP 4 × 180 K microarray (G4890A; Agilent Technologies) following the manufacturer’s protocols. Briefly, 500 ng of purified DNA of a patient and a control (Agilent Technologies) were double-digested with RsaI and AluI enzymes (Agilent Technologies) for 2 h at 37°C. Each digested sample was labeled for 2 h with the Agilent Genomic DNA Labeling Kit, using Cy5-dUTP for the patient DNA and Cy3-dUTP for the control DNA. Labeled products were purified and prepared according to the Agilent protocol. After probe denaturation and pre-annealing with 50 ng of human Cot-1 DNA (Thermo Fisher Scientific, Yokohama, Japan), hybridization was performed at 65°C for 24 h in a rotating oven. After washing steps, the array slide was scanned with an Agilent Microarray Scanner (G2565CA). The spot intensities were measured and the image files quantified using the Agilent Feature Extraction 11.0.1.1 software. Text outputs from the quantitative analyses were imported into Agilent CytoGenomics 4.0 software (Agilent Technologies).

MicroRNA profiling has been described previously in detail [3 (link),4 (link),5 (link)], which we reproduce in quotes as follows: “In brief, total RNA and miRNAs were extracted using the Trizol standard protocol (Invitrogen, Carlsbad, CA, USA) and the mirVANA miRNA isolation kit (Ambion, Austin, TX, USA). Labelling and hybridization were performed using the LabelIT miRNA labelling kit (Mirus Bio LLC, Madison, WI, USA) according to manufacturer’s instructions. Samples were hybridized to Applied MicroArrays (miRlink Bioarray 300054-3PK) platform. This array contained 1211 human miRNAs. Hybridization was performed at 37 °C with rotation at 145 rpm for 16 h. Images were scanned using Agilent Microarray Scanner (G2565CA) controlled by Agilent Scan Control 7.0 software. The total gene signals were extracted using the Imagene 6.0 software (Biodiscovery Inc., El Segundo, CA, USA) that contains summarized signal intensities for each miRNA by combining intensities of replicate probes and background subtraction” [3 (link),4 (link),5 (link)]. In total, 49 CNS tumor samples (the complete cohort described in Section 2.1.) and 13 control samples were investigated for their miRNA expressional profile. All microarray data are MIAME compliant.

Genomic copy number analysis was performed with array-CGH using the Human Genome CGH Microarray kit 105A (Agilent Technologies) following the manufacturer’s recommendations. The target DNA was extracted from peripheral blood using the Wizard Genomic DNA purification kit (PromegaTM, Mannheim, Germany). The DNA control reference was a sex-matched DNA pool recommended by Agilent and provided by Promega (p/n G1521, female; p/n G1471, male). The arrays were scanned at 2 μm resolution using an Agilent microarray scanner (G2565CA) and analyzed using CyoGenomics 3.0 software (Agilent Technologies, Palo Alto, CA, USA). The aberration detection method 2 (ADM-2) algorithm was used to compute and assist in the identification of aberrations for a given sample. Significant chromosomal aberrations were determined using the algorithm ADM-2 with a threshold of 5 and a minimum absolute average log2 ratio of 0.25. Putative chromosome copy number changes were defined by intervals of 3 or more adjacent probes and were considered as being duplicated or deleted when results exceeded |0.25|. All nucleotide positions were based on the Human Reference Sequence Assembly, February 2009 GRCh37/hg19 of the UCSC Genome Browser (http://genome.ucsc.edu/, last access date: 11 November 2022). All the molecular karyotypes were reconstructed following the guidelines of ISCN 2020 [11 ].