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  • br Microarray analysis br As


    2.9. Microarray analysis
    As a primary step, miRNA was isolated after 24 h treatment of the H2DCFDA in a two-step procedure using RT2 qPCR-grade miRNA isolation kit (SABiosciences Corporation, MD, USA) following the manufacturer's instructions. The concentrations of RNA samples were measured by Nanodrop ND1000 (Nanodrop Technologies, USA). Complementary DNAs synthesized through polyadenylataion, elongation, and reverse
    transcription of enriched miRNA samples using the RT2 miRNA first strand kit (SABiosciences). MicroRNA expression profiling for miRNA microarray was performed in duplicates in a 96-well format using the RT2 miRNA PCR array human cancer (SABiosciences) with the RT2 SYBR Green/ROX qPCR master mix (SABiosciences) on the Step One Plus real-time PCR system (Applied Biosystems). Each real-time PCR reaction contained 15 ng of synthesized cDNA, a set of microRNA-spe-cific forward assay primer and universal reverse primer, and 10 µl SYBR green master mix in a total reaction volume of 20 µl. PCR conditions were as follows: 10 min at 95 °C followed by 40 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s. Melting curve analysis was applied to validate whether all primers yield a single PCR product. Relative gene expression levels of each mature miRNA were evaluated using the comparative threshold cycle (Ct) method as normalized to the average Ct value of three housekeeping genes in the array (U6, SNORD47, and SNORD44). Fold change of each miRNA was calculated from the ex-pression levels between the BIBR1532-treated and untreated cells using 2− Ct method in the PCR array data analysis template provided by the manufacturer (SABiosciences).
    2.10. Analysis of gene expression by quantitative real-time PCR
    To carry out qRT-PCR, the cells were treated with BIBR1532 and RNA was extracted by high pure RNA isolation kit (Roche) and quan-tified by a Nanodrop instrument (Nanodrop ND-1000 Technologies). Thereafter, 1 µg of RNA from each sample was applied for reverse transcription using the revertAid First Strand cDNA synthesis kit (Takara BIO, Japan). The prepared cDNA was subjected to qRT-PCR using SYBR Premix Ex Taq technology (Takara BIO) on a light cycler instrument (Roche). Thermal cycling conditions were an initial acti-vation step of 30 s at 95 °C followed by 40 cycles including a dena-turation step of 15 s at 95 °C and a combined annealing/extension step of 60 s at 60 °C. Melting curves were analyzed to validate single PCR product of each primer, and the values for the relative quantification were calculated based on 2− ct relative expression formula. Nucleotide sequences of the primers used for qRT-PCR were listed in Table 1.
    2.11. Bioinformatics analysis
    The probable biological functions of dysregulated miRNAs (P value < 0.05) in response to BIBR1532 were evaluated by performing validated analysis using miRTarBase computational programs. To ca-tegorize the miRNA targets based on their involvement in biological processes, cellular components and molecular functions, we applied GO term analysis using DAVID (Database for Annotation, Visualization and Integrated Discovery) software ( jsp). Moreover, DIANA miRPathv3.0 was applied to produce heatmap. The biochemical functions of the miRNAs, and their deregulation in various malignancies, were further defined by Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis using DAVID online database, which is a comprehensive set of functional annotation tools used for
    Table 1 r> Nucleotide sequences of primers used for real-time RT-PCR. 
    understanding the biological meaning behind large list of genes. P va-lues of each pathway were accustomed by the method of Benjamini-Hochberg to control the false discovery rate (FDR). In the present study, GO terms and signaling pathways were selected with the threshold of significance being defined as P < 0.01 and FDR < 0.05. Using miRTarBase computational programs, we found validated target genes for BIBR1532-dysregulated miRNAs. The integrations between altered miRNAs and validated target gene regulatory networks were assessed by Cytoscape software.
    2.12. Statistical analysis
    Experimental data are expressed by mean ± standard deviation (S.D.) of three independent assays. All tests were done in duplicate or triplicate. An independent t-test was conducted for comparison between the expressions of miRNAs in the BIBR1532-treated and untreated cells. Statistical significance was calculated using paired two-tailed Student's t-tests. Statistically different values were defined significant at *, P ≤ 0.05.
    3. Results
    3.1. BIBR1532 exerted cytotoxic effect on cancer cell lines