Genepix 4

Fluorescent aRNA targets were prepared from 1 µg total RNA samples using OneArray^® Amino Allyl aRNA Amplification kit (Phalanx Biotech Group) and Cy5 dye (GE Healthcare Life Sciences). Fluorescent targets were hybridized to the Mouse Whole Genome OneArray^® with Phalanx hybridization buffer using Phalanx Hybridization system. After 16 h hybridization, non-specific binding targets were removed using saline and sodium citrate buffer. The slides were scanned using a DNA Microarray Scanner (Model G2505C; Agilent Technologies, Inc.). The Cy5 fluorescent intensities of each spot were analyzed using GenePix 4.1 software (Molecular Devices, LLC, Sunnyvale,. CA, USA). Each single sample was at least assessed twice in terms of technical or biological replicates under the reproducibility >0.975. The signal intensity was loaded into Rosetta Resolver system^® (Rosetta Biosoftware, Seattle, WA, USA) for data pre-processing and 75 percentile centering normalization was applied. The errors of the sample were estimated by using the error-weighted approach at the same time. The fold change and P-value for pair-wise sample comparison were calculated for evaluating differentially expressed genes (DEGs). The criteria with log₂|fold change|≥0.5 and P<0.05 were used for further analysis.

Total RNA was extracted from cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Small RNA was pre-enriched by Nanoseplook (Pall Corporation, Port Washington, NY, USA) from 2.5 µg total RNA samples and labeled with miRNA ULS™ Labeling Kit (Kreatech Diagnostics; Leica Biosystems St. Louis, GmbH, Wetzlar, Germany). Labeled miRNA targets were hybridized to the Human miRNA OneArray^® v3 with OneArray^® Hybridization System. Subsequent to 16 h hybridization at 37°C, non-specific binding targets were washed away by three different washing steps: Wash I, 37°C for 5 min; Wash II, 37°C for 5 min, then 25°C for 5 min; Wash III, rinse 20 times), and the slides were dried by centrifugation at 671 × g and room temperature for 1 min and scanned by an Axon 4000B scanner (Molecular Devices, LLC). The Cy5 fluorescent intensities of each probe were analyzed by GenePix 4.1 software (Molecular Devices).
The raw intensity of each probe was processed by R program (version v2.12.1; https://www.r-project.org). Probes that passed the criteria were normalized by 75% median scaling normalization method. Normalized spot intensities were transformed to gene expression log₂ ratios between the control and treatment groups. The spots with log₂ ratio ≥1 or log₂ ratio ≤-1 and P<0.05 were tested for additional analysis.