G2565ca microarray scanner system

Total RNA was extracted from 250 μL of plasma using the TRIzol reagent according to the manufacturer’s instructions (Invitrogen) and purified using an RNeasy minikit (Qiagen). To increase RNA recovery, 1 μg of MS2 carrier RNA was added to each plasma sample. The quality of the total RNA isolated was determined using an Agilent 2100 Bioanalyser.
The plasma miRNA expression levels for 24 individuals were assessed using SurePrint G3 human 8 × 60 k miRNA microarrays (Agilent Technologies), covering 1205 human miRNAs (Sanger miRBase release 16). The miRNAs were dephosphorylated and labelled with cyanine 3-cytidine biphosphate, including a labelling spike-in solution (Agilent Technologies) to assess the labelling efficiency. The samples were hybridized on the arrays, including a hybridization spike-in solution (Agilent Technologies) to monitor the hybridization efficiency. The arrays were scanned with a G2565CA Microarray Scanner System with SureScan High Resolution Technology (Agilent Technologies) using Scan Control software. The Feature Extraction 11.5.11 (Agilent Technologies) and GeneSpring 12.6.1 software packages were used for data processing, and all steps were performed according to the manufacturer’s protocol.

Data files from the Agilent G2565CA Microarray Scanner System were analyzed using the GeneSpring (Agilent Technologies, United States, version 12.5) as described previously (Kwiatek et al., 2014 (link), 2015 (link)). Variations were presented as the ratio of gene expression of wt gonococcal strain over-expression of the same gene for the NgoAV knockout mutant (strain NgoΔAV: ngoAVhsdS1::cm). Genes with different expressions at least 2.0-fold and a p-value < 0.05 were further analyzed. Microarray data have been deposited in the National Center for Biotechnology Information (NCBI) and are accessible through Gene Expression Omnibus series under the accession number GSE71703 (Edgar et al., 2002 (link)).

Data files from the Agilent G2565CA Microarray Scanner System were loaded into the GeneSpring (Agilent Technologies, version 12.5) and analyzed as described previously (Kwiatek et al., 2014 (link)). Variations were presented as the ratio of gene expression of wt gonococcal strain over expression of the same gene for the NgoAXP knock-out mutant. Genes with divergent expression of ≥1.5 or 2-fold and a P-value < 0.05 were chosen. Microarray data have been deposited in the NCBI’s and are accessible through Gene Expression Omnibus series accession number GSE71703¹ (Edgar et al., 2002 (link)).

Karyotyping based on R or G banding was performed using standard methods on metaphase spreads from primary cell lines, MSC and OSDC of the patients.
Genomic DNAs were extracted from primary cell lines, MSC, and OSDC using standard protocols. Array comparative genomic hybridization (aCGH) experiments were performed by using Agilent Human Genome CGH 400K oligonucleotide arrays or 60K oligonucleotide arrays with the ISCA design (Agilent, Santa Clara, CA, USA; Available online: www.agilent.com) following the protocols provided by Agilent. Arrays were scanned with G2565CA Microarray Scanner System and analyzed with CytoGenomics v3.0.6.6 (Agilent Technologies, Santa Clara, CA, USA).

aCGH analyses were carried out using the oligonucleotide-based SurePrint G3 Human Genome CGH + SNP Microarray kit 4 × 180 K (Agilent Technologies, CA, USA), as described by the manufacturer instructions. Briefly, 1 µg of reference DNA (Agilent Euro Male/Female) and patient DNA were digested and labeled using the SureTag DNA Labeling kit (Agilent Technologies). Fragmented DNAs were labeled with Cy3 (reference DNA) and Cy5 (test samples) fluorescent dUTP, respectively. Purification columns were used to remove the unincorporated nucleotides and dyes.
After purification, labeled sample and reference DNA were co-hybridized at 65 °C at 20 rpm for 24 h on the array slides, and scanned with Agilent G2565CA Microarray Scanner System. Features were normalized and extracted using feature extraction software (v11.1). Data were analyzed and visualized by Cytogenomics software (v2.7). For calling genomic imbalances, we applied the statistical algorithm ADM-2 with a sensitivity threshold of 6.0. A minimum of three-probe for aberration was used for filter. The log2 ratio of Cy3:Cy5 signal intensities was used by the program to consider a variation as a loss or a gain, with reasons ≤ −0.25 reasons considered loss. The log2 ratio of < −1.0 at the region of interest was considered to represent a homozygous deletion.