Benzo[a]pyrene-induced changes in microRNA-mRNA networks
ABSTRACT: The microarray contains 9360 reporters, each present four times on the microarray. In our analysis 1326 human and viral reporters were used, of which 880 are unique human mature microRNAs. The other 8024 reporters represent microRNAs from other species. cDNA was generated using 1 μg of total RNA per sample. RNA was labeled only with Hy3 containing dye using the microRNACURY LNA™ microRNA Hy3 Power labeling kit (Exiqon, Denmark) according to the manufacturer’s protocol in a total volume of 25 μl. To adjust the volume to 100 μl, a hybridization buffer provided in miRCURY LNA™ microRNA Array, 5th generation kit (Exiqon, Denmark) was used. For each biological experiment, one hybridization per time point was conducted and one sample per array. In total, 24 arrays were used for 8 different conditions (BaP or control, at 6, 12, 24 and 48 hours).
Project description:The microarray contains 9360 reporters, each present four times on the microarray. In our analysis 1326 human and viral reporters were used, of which 880 are unique human mature microRNAs. The other 8024 reporters represent microRNAs from other species. cDNA was generated using 1 μg of total RNA per sample. RNA was labeled only with Hy3 containing dye using the microRNACURY LNA™ microRNA Hy3 Power labeling kit (Exiqon, Denmark) according to the manufacturer’s protocol in a total volume of 25 μl. To adjust the volume to 100 μl, a hybridization buffer provided in miRCURY LNA™ microRNA Array, 5th generation kit (Exiqon, Denmark) was used. Overall design: For each biological experiment, one hybridization per time point was conducted and one sample per array. In total, 24 arrays were used for 8 different conditions (BaP or control, at 6, 12, 24 and 48 hours).
Project description:Sixth generation Exiqon® locked nucleic acid miRCURY™ LNA microarrays were used to search and validate some unidentified miRNAs that regulate EMT in head and neck cancer carcinoma. MiRNA array screening was performed to identify the differential expression of miRNAs involved in EMT in natural epithelial - mesenchymal phenotype cell line pair（HN-4, HN-12) and in TGF-β induced EMT models (HN-4 TGF-β,HN-4). HN-4 parental cell was served as the control.One µg total RNA from sample and control was labeled with Hy5™ and Hy3™ fluorescent label, respectively, using the miRCURY™ LNA Array power labeling kit (Exiqon, Denmark) following the procedure described by the manufacturer.
Project description:Cells of a human cholangiocyte cell line (H69) were exposed to 10 ng/ml of IFN-gamma for 8 h. Cells were collected and processed for miRNA array analysis carried out by EXIQON (Denmark).
Project description:The representative cell line, HT29, culture in hypoxic and normoxic conditions were used to perform the miRNA microarray. The chip assay and data analysis were entrusted to KangChen Bio-tech, Inc. (Shanghai, China) and all the miRNAs with more than 2-fold variation in different samples were identified. Detailed methods were performed as previously described24 using the miRCURY Hy3 labeling kit and the microRNA array software (Exiqon, Denmark).
Project description:Total RNA was extracted from adipose tissue of high (full) fat diet and standard fat diet mice. Adipose tissue was taken from 16 mice in total. Eight mice were fed a standard diet (SD; control) (10 kcal% fat) and the other eight were fed a high-fat diet (HFD; test) (60 kcal% fat; Research Diets, New Brunswick, NJ) for 5 months. Total RNA was isolated from pooled White Adipose Tissue from SD- or HFD-fed mice using guanidinium thiocyanate. The quality of the total RNA was verified by an Agilent 2100 Bioanalyzer profile. One µg total RNA from sample and reference were labeled with Hy3™ and Hy5™ fluorescent label, respectively, using the miRCURY™ LNA Array power labeling kit (Exiqon, Denmark) following the procedure described by the manufacturer. The Hy3™-labeled samples and the Hy5™-labeled sample were mixed pair-wise and hybridized to the miRCURY™ LNA array version 10.0 (Exiqon, Denmark), which contains capture probes targeting all miRNAs for all species registered in the miRBASE version 11.0 at the Sanger Institute. The hybridization was performed according to the miRCURY™ LNA array manual using a Tecan HS4800 hybridization station (Tecan, Austria). After hybridization the microarray slides were scanned and stored in an ozone free environment (ozone level below 2.0 ppb) in order to prevent potential bleaching of the fluorescent dyes. The miRCURY™ LNA array microarray slides were scanned using the Agilent G2565BA Microarray Scanner System (Agilent Technologies, Inc., USA) and the image analysis was carried out using the ImaGene 8.0 software (BioDiscovery, Inc., USA). The quantified signals were normalized using the global Lowess (LOcally WEighted Scatterplot Smoothing) regression algorithm.
Project description:Fifth generation Exiqon® locked nucleic acid miRCURY™ LNA microarrays were used to profile the expression of microRNAs in whole blood of patients with non-small cell lung adenocarcinoma and of clinically relevant controls without the disease. Total RNA was isolated from whole blood of 22 cases and 23 controls. Sample RNA was labeled with the Cy3-like Hy3™ dye, mixed with a human universal reference RNA labeled with the Cy5-like Hy5™ dye, and hybridized to two-color miRCURY™ arrays from Exiqon®. - Disease: Lung adenocarcinoma: samples 1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 16, 17, 18, 20, 21, 22, 23, 24, 25 - Disease: No cancer: samples 26, 27, 28, 29, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49
Project description:We compared time-series miRNA expression profile in high-phosphate–stimulated rat vascular smooth muscle cells at 0, 3, and 12 hr respectively. Overall design: Total RNA of rat VSMCs stimulated with 2.6 mM inorganic phosphate for 0, 3 or 12 hr was harvested by the Trizol method (Invitrogen) or use of the miRNeasy mini kit (QIAGEN). The samples were labeled by use of the miRCURY™ Hy3™/Hy5™ Power labeling kit and hybridized on the miRCURY™ LNA Array (v.16.0) (Exiqon, Denmark). After washing slides were scanned by use of the Axon GenePix 4000B microarray scanner. Expressed data were normalized by the Median normalization method, and then relative fold change at each time was detected by level of fluorescence in the phosphate-treated group normalized to that in the control group. Finally, hierarchical clustering was performed for miRNA expression profiling. Student t test was used to compare differences between 2 groups, and a 1.5-fold change was considered significant.
Project description:Comparing miRNAs expression levels in chorioamniotic membranes from women at term in labor (TL), women at term not in labor (TNL) and women who deliverd preterm (PTLC). The goal was to see if miRNA levels are indicators of preterm delivery or spontaneous labor at term. A two-channel technology was used in this experiment in which a pooled reference RNA was used for competitive hybridization. The pooled reference was generated at Exiqon in Denmark from a mixture of several human tissues (placenta, thyroid, brain, adipose, spleen, liver, colon, skeletal muscle, ovary, kidney, heart, cervix, testes, esophagus, small intestine, prostate, trachea, thymus, bladder, lung).
Project description:The well-defined battery of in vitro systems applied within chemical cancer risk assessment is often characterised by a high false-positive rate, thus repeatedly failing to correctly predict the in vivo genotoxic and carcinogenic properties of test compounds. Toxicogenomics, i.e. mRNA-profiling, has been proven successful in improving the prediction of genotoxicity in vivo and the understanding of underlying mechanisms. Recently, microRNAs have been discovered as post-transcriptional regulators of mRNAs. It is thus hypothesised that using microRNA response-patterns may further improve current prediction methods. This study aimed at predicting genotoxicity and non-genotoxic carcinogenicity in vivo, by comparing microRNA- and mRNA-based profiles, using a frequently applied in vitro liver model and exposing this to a range of well-chosen prototypical carcinogens. Primary mouse hepatocytes (PMH) were treated for 24 and 48h with 21 chemical compounds [genotoxins (GTX) vs. non-genotoxins (NGTX) and non-genotoxic carcinogens (NGTX-C) versus non-carcinogens (NC)]. MicroRNA and mRNA expression changes were analysed by means of Exiqon and Affymetrix microarray-platforms, respectively. Classification was performed by using Prediction Analysis for Microarrays (PAM). Compounds were randomly assigned to training and validation sets (repeated 10 times). Before prediction analysis, pre-selection of microRNAs and mRNAs was performed by using a leave-one-out t-test. No microRNAs could be identified that accurately predicted genotoxicity or non-genotoxic carcinogenicity in vivo. However, mRNAs could be detected which appeared reliable in predicting genotoxicity in vivo after 24h (7 genes) and 48h (2 genes) of exposure (accuracy: 90% and 93%, sensitivity: 65% and 75%, specificity: 100% and 100%). Tributylinoxide and para-Cresidine were misclassified. Also, mRNAs were identified capable of classifying NGTX-C after 24h (5 genes) as well as after 48h (3 genes) of treatment (accuracy: 78% and 88%, sensitivity: 83% and 83%, specificity: 75% and 93%). Wy-14,643, phenobarbital and ampicillin trihydrate were misclassified. We conclude that genotoxicity and non-genotoxic carcinogenicity probably cannot be accurately predicted based on microRNA profiles. Overall, transcript-based prediction analyses appeared to clearly outperform microRNA-based analyses. The study investigated differential gene expression in primary mouse hepatocyte mRNA following 24 and 48 hours of exposure to di(2-ethylhexyl)phthalate(DEHP), 4-Acetylaminofluorene(4AAF), Curcumin(Cur), Phenobarbital(PhB),Reserpine(Res), 7,12-Dimethylbenzanthracene (DMBA), Resorcinol(Resorcinol), Para-cresidine(pCres), Phenacetin(Phen), Diclofenac(diclo),Wy 14643(Wy), Tributyltinoxide(TBTO), Benzo[a]pyrene(BaP), 8-Hydroxyquinoline [AKA 8-quinolinol](8HQ), 17-b-estradiol(E2), ampicillin(AmpC), cisplatin(CisPl), Aflatoxin B1(AFB1), Cyclosporin A(CsA), 2,3,7,8-Tetrachlorodibenzo-p-dioxin(TCDD), Quercetin(Que) or their responsive solvent (dimethylsulfoxide(DMSO), Ethanol(ETOH), phosphate buffered saline(PBS)). Three biological replicates per compound/solvent. In total 184 arrays.
Project description:HepG2 cells (human liver hepatocarcinoma) were exposed for 6 different time points (6,12,18,24,36 and 48h) to Benzo[a]pyrene (BaP) in duplicate. Total RNAs were ribo-depleted and sequenced on an Illumina Hiseq2000 in pair-end 100 bp reads. One of the duplicates for BaP-treated sample at the 24 hour time point was found to have a minor fastq file integrity issue and was removed from this data set as a precaution.