Mircury lna array system

The microarray analysis for miRNA profiling was conducted by the Shanghai Kangcheng Technology using the miRCURY LNA Array system (Exiqon, Vedbaek, Denmark). Total RNA was extracted and purified using the mirVana miRNA Isolation Kit (Ambion, Austin, TX, USA) following the manufacturer's instructions. The miRCURY™ Hy3™/Hy5™ Power labeling kit (Exiqon, Vedbaek, Denmark) was used according to the manufacturer's guideline for miRNA labeling. After stopping the labeling procedure, the Hy3™-labeled samples were hybridized on the miRCURYTM LNA Array (v.19.0) (Exiqon) according to array manual. Following hybridization, the slides were achieved, washed several times using Wash buffer kit (Exiqon). Expressed data were normalized using the Median normalization. After normalization, significant differentially expressed miRNAs between two groups were identified through Fold change (>2) and P-value (<0.05).

The microarray analysis for miRNA profiling was conducted by the miRCURY LNA Array system (Exiqon, Vedbaek, Denmark). The total RNA was extracted from intestinal samples of mice subjected to intestinal I/R or sham surgery using Trizol (Invitrogen) according to the instructions provided by the manufacturer. The quality and quantity of RNA samples were assessed by using nanodrop spectrophotometer (ND-1000, Nanodrop Technologies). RNA samples were labeled with an Exiqon miRCURY™ Hy3™/Hy5™ Power labeling kit and hybridized on a miRCURYTM LNA Array station. Scanning was performed with an Axon GenePix 4000B microarray scanner (Axon Instruments, Foster City, CA). Scanned images were then imported into GenePix Pro 6.0 software (Axon) for grid alignment and data extraction. Replicated miRNAs were averaged and miRNAs that intensities ≥30 in all samples were chosen for calculating normalization factor. Expressed data were normalized using the Median normalization. Discriminant miRNAs and differences between groups were analyzed using Bayes moderated t-test (limma) with Benjamini Hochberg false discovery rate at P < 0.05, unless otherwise specified. A two-fold cut-off was applied to select up-regulated and down-regulated miRNAs.

The human lung adenocarcinoma cell line A549 (American Type Culture Collection) was maintained in Dulbecco's modified Eagle's medium (DMEM) at 37°C in 5% CO₂. Cells were seeded in 10 cm culture dishes and exposed to 500 μg/mL PM_2.5 with three biological replicates. Control groups were treated with culture medium. Total RNA was extracted with the TRIZOL reagent (Invitrogen, 15596-026, USA) according to the manufacturer's instructions.
The microarray analysis for miRNA profiling was conducted by the miRCURY LNA Array system (Exiqon, Vedbaek, Denmark). Raw data were subjected to background subtraction and normalization with the limma R-package [43 (link)]. Discriminant miRNAs and differences between groups were analyzed using Bayes moderated t-test (limma) with Benjamini Hochberg false discovery rate (FDR) at P < 0.05, unless otherwise specified. A two fold cut-off was applied to select up-regulated and down-regulated miRNAs.

Total cellular RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and cDNAs were synthesized using the ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan). Real-time PCR was performed using SYBR Green Realtime PCR Master Mix (Roche, Basel, Switzerland) and the ABI ViiA7 QPCR System (Applied Biosystems).
To evaluate the differentially expressed miRNAs in AZ505-treated and siSMYD2-transfected RCC cells relative to control cells, siSMYD- (n = 4), AZ505- (n = 4), and negative control (n = 4)-treated 786-O cells were subjected to miRNA microarray analysis by Kangcheng Technology (Shanghai, China), using the miRCURY LNA Array system (Exiqon, Vedbæk, Denmark). The cutoff for defining candidate downregulated miRNAs was fold-change < 0.5 and P < 0.05.
ChIP was performed using the EZ-Chromatin Immunoprecipitation Kit (Millipore, Billerica, MA, USA, #17-371) according to the manufacturer's instructions. The DNA fragments were then extracted and analyzed by PCR using the following primer pair specific for the miR-125b promoter: Forward, 5′-TCCTTGAGAGCAACACGCAG-3′ and reverse, 5′-GTGGAGTTTGAAAGTTGGAG-3′.