Agilent sureprint g3 human v2 ge 8 60 k microarray

MDA-MB-231-P and MDA-MB-231-IV2-1 cells were cultured in DMEM supplemented with 10% inactivated FBS. The cells were subjected to RNA extraction after 70% confluence was reached. Breast cancer cell total RNA was prepared using TRIZOL approach. Next, 0.2 μg of total RNA was subjected to amplification by using a Low Input Quick-Amp Labeling kit (Agilent Technologies, United States) and labeled with Cy3 (CyDye, Agilent Technologies, United States). Finally, 0.6 μg of Cy3-labled cRNA was fragmented and hybridized using an Agilent SurePrint G3 Human V2 GE 8 × 60K microarray (Agilent Technologies, United States) at 65°C for 17 h. After hybridization, the microarray image was scanned and analyzed using Feature Extraction 10.5.1.1 (Agilent Technologies, United States). We performed microarray experiments by using the Agilent oligonucleotide ChIP-on-chip protocol, then data analysis were analyzing by Welgene Biotech (Taipei, Taiwan) We submitted all microarray raw data to the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO), and they are freely available (accession number: GSE175513).

The expression profiles of mRNAs in Jurkat cells cocultured with culture medium or BDNF 200 ng/mL for 48 h were evaluated using microarray analysis. The microarray analysis was performed by Welgene Biotech (Taipei, Taiwan) as in our previous study [38 (link)]. In brief, total RNA was extracted by TRIzol reagent (Invitrogen, Carlsbad, CA, USA), then quantified at OD_260nm by an ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and quantified using a Bioanalyzer 2100 (Agilent Technology, USA) with an RNA 6000 lab chip kit (Agilent Technologies, Santa Clara, CA, USA). Total RNA was labeled with cyanine 3 (Cy3; Agilent Technologies) dye and hybridized to Agilent SurePrint G3 human V2 GE 8 × 60 K microarray (Agilent Technologies). Microarrays were scanned with an Agilent microarray scanner (Agilent Technologies), and the scanned images were analyzed by Feature extraction 10.5.1.1 software (Agilent Technologies, USA); image analysis and normalization software were used to quantify signal and background intensity for each feature. Raw signal data were normalized by quantile normalization for differential expressed genes discovering.

To profile the expression of NCMT and HNSCC, total tissue RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA), and then the RNeasy Mini Kit (Qiagen, Hilden, Germany) was used. The isolated RNA was quantified at OD 260 nm and qualitatively analyzed using a bioanalyzer (Agilent Technology, USA). A 0.2 μg mass of total RNA was amplified using a Low-Input Quick-Amp Labeling Kit (Agilent Technologies, USA) and labeled with Cy3 (CyDye, Agilent Technologies, USA) during an in vitro transcription process. A 0.6 μg mass of Cy3-labled cRNA was fragmented to a mean size of approximately 50-100 nucleotides by incubation with a fragmentation buffer at 60°C for 30 minutes. Similarly fragmented labeled cRNA is then pooled and hybridized using an Agilent Sure Print G3 Human V2 GE 8 × 60 K Microarray (Agilent Technologies, USA) at 65°C for 17 h. After the microarrays are washed and dried by blowing with a nitrogen gun, they are scanned using an Agilent microarray scanner (Agilent Technologies, USA) at 535 nm for Cy3. Scanned images are analyzed using Feature Extraction 10.5.1.1 software (Agilent Technologies, USA) for image analysis and normalization to quantify the signal and background intensity for each feature (Welgene Biotech. Co., Taipei, Taiwan).