CDNA microarray analysis of miR-1 transfected HeLa and NPC-TW01 cells
ABSTRACT: To investigate the mechanisms of miR-1 inducing apoptosis, we performed cDNA expression microarray analysis to identify the candidate miR-1 regulating genes that are involved in apoptosis. Overall design: NPC-TW01 cells were transfected with miR-1 or miR-negative control for 40 hours, their total RNA were isolated from the cells and hybridized to an Agilent human whole genome oligo 4 x 44 K microarray. HeLa cells were transfected with miR-1 or miR-negative control for 40 hours, their total RNA were isolated from the cells and hybridized to an Agilent human whole genome oligo 4 x 44 K microarray.
INSTRUMENT(S): Agilent-014850 Whole Human Genome Microarray 4x44K G4112F (Feature Number version)
Project description:To investigate the mechanisms of miR-1 inducing apoptosis, we performed cDNA expression microarray analysis to identify the candidate miR-1 regulating genes that are involved in apoptosis. NPC-TW01 cells were transfected with miR-1 or miR-negative control for 40 hours, their total RNA were isolated from the cells and hybridized to an Agilent human whole genome oligo 4 x 44 K microarray. HeLa cells were transfected with miR-1 or miR-negative control for 40 hours, their total RNA were isolated from the cells and hybridized to an Agilent human whole genome oligo 4 x 44 K microarray.
Project description:Nasopharyngeal carcinoma (NPC) is endemic in Southeast Asia and southern China. The primary treatment for NPC is radiotherapy. Despite of the encouraging results of radiotherapy, local recurrence or distant metastases of NPC after the initial therapy is frequently found due to radioresistance. Therefore, there is an urgent need to identify genes that control radiosensitivty, aiming to reduce disease recurrence. Epstein-Barr virus (EBV) infection was closely associated with undifferentiated NPC. EBV-encoded microRNAs (miRNAs) played crucial roles in the pathogenesis of NPC. Ebv-miR-BART7 belongs to the 44 EBV BART miRNAs and was found to be up-regulated in NPC tissues and plasma. Forced expression of ebv-miR-BART7 enhanced the radiosensitivity of NPC cells. However, the mechanisms underlying the sensitizing effect of ebv-miR-BART7 on radiation remain largely unknown. Given that miRNA exerts biological functions by regulating its targets, we used microarray to identify the targets of ebv-miR-BART7 that regulate radiosensitivity of NPC cells. Overall design: NPC HONE1 cells were transfected with ebv-miR-BART7 mimic or negative control siRNA. The experiment was done in duplicate. Then, HONE1 cells were irradiated at 4 gray. RNA were extracted and hybridized on Affymetrix microarrays.
Project description:To explain the mechanism that miR-29c affects the cell proliferation, we attempted to identify the miR-29c target genes in gastric carcinoma. The expression profiles in MKN45, MKN7 and MKN74 cells transfected with miR-29c oligo or Negative control oligo were obtained from microarray analysis. Then, the genes differentially expressed (Fold change >= 2.0) in miR-29c-transfected cells compared with negative control-transfected ones were identified in each cell lines, respectively. The differentially expressed genes shared among 3 cell lines were identified as the candidates for miR-29c targets. Human gastric cancer cell lines, MKN45, MKN74 and MKN7 were transfected with miR-29c oligo or negative control oligo (n=2) (Ambion). At 24h after, total RNA was extracted and microarray analysis was performed. The genes with common expression changes among three cell lines miR-29c-transfected were identified as the candidates for miR-29c targets.
Project description:In order to identify direct or indirect miR-27a modulated genes, RH36 cells transfected with miR-27a precursors (pre-miR-27a or pre-control), analyzed 48h post-transfection, were used to perform microarray analysis. Gene expression profiling was carried out in ERMS cell line (RH36) transfected with miR-27a or with a negative pre-control. We performed the experiments in triplicate. We used the ‘‘Whole Human Genome Oligo Microarray’’ (Agilent), consisting of 41.000 (60-mer) oligonucleotide probes, which span conserved exons across the transcripts of the targeted full-length genes.
Project description:We have employed whole genome microarray expression profiling as a discovery platform to screen potential target genes of miR-204-5p in colorectal cancer cell HCT116. HCT116 cells were seeded in 6-cm2 tissue culture plates and transfected with the miR-204-5p mimic or negative control (NC) using Lipofectamine 2000 (Invitrogen, USA). After propagation for 48 hours, total RNA was extracted using TRIzol reagent (Invitrogen, USA). Expression profiling was performed using an Agilent human whole genome oligo microarray chip (4×44K) (Agilent, USA) Expression profiling in HCT116 cells was measured at 48 hours after transfection with miR-204-5p or negative control. Experiments were performed using the two samples without repeat experiment.
Project description:To identify which miR-148b targets were involved in tumorigenesis, a microarray analysis was performed for miR-148b over-expressing cells versus controls and 129 (49 up and 80 down) modulated genes were revealed. The effects of miR-148b on cancer progression depend on the direct and indirect regulation of multiple target genes. To identify miR-148b modulated genes, MDA-MB-231 cells were transfected with miR-148b precursors or negative controls (pre-miR-148b or control) and used 48h later for microarray and western blot (WB) analyses. When a “Whole Human Genome Oligo Microarray” (Agilent) platform was employed, 129 differentially expressed genes (49 upregulated, 80 downmodulated) were found at 48h, considering a fold change (FC) cut of 1.5 and a false discovery rate (FDR) of 16% (Table S4). Crossing these results with the list of putative miR-148b targets (3642) obtained by the miRecords System, we observed that 33 of the modulated genes were also miR-148b predicted targets and interestingly, 26 out of these 33 genes were downmodulated.
Project description:Gene expression profiling of MCF7 cells transfected with a miR-139-5p or negative control mimic Overall design: MCF7 cells were transfected with a miR-139-5p or negative control mimic. Total RNA was extracted 24 hours later and analyzed by Agilent gene expression microarray
Project description:To investigate the gene expression in human corneal epithelial overexpressing hsa-miR-145 by transfection , we have employed Whole Human Genome Oligo Microarray (Agilent) as a screening platform to identify gene regulation. We discovered a differential gene expression in HCE cells transfected with hsa-mIR-145 against cells with scrambled sequences. Among them, genes related with corneal development, integrity, differentiation and inflammatory responses were found and this was validated by real-time PCR. Overall design: HCE cells transfected with hsa-miR-145 or scrambled sequences were collected at 24 hours after transfection. Total RNA was extracted by Trizol/chloroform and purified with RNeasy mini spin column. RNA samples with 28S/18S ratios in the range of 1.4 to 1.8 were used for expression RNA profiling using Whole Human Genome Oligo Microarray (Agilent).
Project description:Compulsory expression of miR-210 in normal endometrial stromal cells directed the induction of cell proliferation and vascular endothelial growth factor production, and the inhibition of apoptosis in through signal transducer and activator of transcription 3 (STAT3) activation. Accumulating evidence suggests that microRNAs play definite roles in the pathogenesis of endometriosis. The objective of the study was to determine the role of miR-210, one of the upregulated microRNA in endometriotic cyst stromal cells, in the pathogenesis of endometriosis. Downstream targets of miR-210 were identified by Compulsory expression of miR-210 in normal eutopic endometrial stromal cells, a global mRNA microarray technique, and Ingenuity pathways analysis. NESCs were transfected with precursor hsa-miR-210 (Pre-miRTM miRNA precursor- hsa-miR-210, Ambion, Austin, TX, USA) or negative control precursor miRNA (Pre-miRTM miRNA precursor-negative control #1 Ambion) at a final concentration of 10 nM, using LipofectamineTM RNAiMAX (Invitrogen, Carlsbad, CA, USA). Forty-eight hours after transfection, total RNA from cultured NESCs transfected with precursor hsa-miR-210 (n=4) and NESCs (n=4) transfected with negative control precursor miRNA was extracted with an RNeasy Mini kit (Qiagen, Valencia, CA, USA). Then, the samples were subjected to a gene expression microarray analysis with a commercially available human mRNA microarray (G4845A, Human Gene Expression 4x44K v2, Agilent Technologies, Santa Clara, CA, USA).
Project description:To identify genes affected by miR-634 overexpression, we transfected with 20nmol of miR-634 or miR-negative control (NC) in HeLa, KYSE850, or U2OS cells. After 2 days, RNA was extracted, and then expression analysis was performed using agilent microarray. Expression microarray with miR-634 or miR-NC transfected HeLa, KYSE850, or U2OS cells were performed in duplicate.