Sureprint g3 human cgh microarray 8 60k

Sample preparation, slide hybridization, and analysis were performed using SurePrint G3 Human CGH Microarray 8×60K (Agilent, Santa Clara, CA) according to the manufacturer’s instructions. Sex-matched commercial DNA samples (Promega) were used as reference DNA during aCGH. The arrays were scanned at 2-mm resolution using Agilent microarray scanner and analyzed using Feature Extraction v10.7 and Agilent Genomic Workbench v5.0 softwares. The Aberration Detection Method 2 (ADM2) algorithm prompted by Genomic Workbench software was used to compute and assist the identification of aberrations for a given sample (threshold = 5; log2 ratio = 0.3). To calculate the estimated percentage of mosaicism we used the formula determined by Cheung et al. [26 (link)].

We performed karyotyping on peripheral blood lymphocytes of the index patient using the Giemsa-banding technique at 550 band resolution per haploid genome. Moreover, array comparative genomic hybridization (aCGH) (SurePrint G3 Human CGH Microarray 8×60k; Agilent Technologies) was performed on the high-quality genomic DNA (gDNA) of the index. gDNA was extracted from the peripheral blood lymphocytes using the MagCore® HF16 automated nucleic acid extractor (RBC Bioscience Corp.).

For each experiment, purified FFPE DNA (500 ng) and reference DNA (500 ng) (NA10851; Coriell Institute for Medical Research, Camden, NJ, USA) were differentially labeled with cyanine 5 (Cy5) and cyanine 3 (Cy3) fluorescent dyes using Universal Linkage System technology (Agilent Technologies), a non-enzymatic direct labeling method. Unreacted dye was removed using KREApure purification columns (Agilent Technologies). DNA labeling efficiency was assessed by NanoDrop 2000 spectrophotometry (ND-2000; Thermo Fisher Scientific, Waltham, MA, USA). The degree of labeling (DoL, the number of fluorophore molecules per 100 nucleotides) was calculated using post-labeling DNA yield and fluorophore concentration. DoL values ranging from 0.75% to 2.5% and 1.75% to 3.5% were considered optimal for Cy5- and Cy3-labeled DNA, respectively. Cy5- and Cy3-labeled DNA hybridization was performed using dual-color array containing 60-mer oligonucleotide probes (SurePrint G3 Human CGH Microarray 8 × 60 K; Agilent Technologies). Following hybridization, the arrays were scanned using a Dual Laser Microarray Scanner (G2565CA; Agilent Technologies). The images were extracted and analyzed using Feature Extraction software version 10.5.1.1 (Agilent Technologies) and DNA Analytics software version 4.0.73 (Agilent Technologies).