ABSTRACT: To gain more insight in the expression and role of microRNAs in trophoblastic cells, we have assessed the miRNA expression pattern of four trophoblastic cell lines and have compared them with the miRNA signature of human isolated 3rd trimester cytotrophoblast cells. Total RNA was reverse-transcribed, pre-amplified with miRNA-specific primers, and loaded into TLDA cards. Quantitative real-time PCR was performed for 768 miRNAs. For each of two cards per sample (A and B), 9 ul of pre-amplified diluted sample was mixed with TaqMan reaction mix (Applied Biosystems) and loaded into miRNA TLDA cards (Applied Biosystems) by centrifugation. Cards were sealed, and qRT-PCR was performed at a real-time thermocycler (ABI-7900, Applied Biosystems) following the manufacturer's recommendations. MiRNA signatures of human primary 3rd trimester trophoblast cells were compared with those of four trophoblastic cell lines
Project description:miRNA expression profiling was performed on MM.1S MM cells cultured 8 hours in control media or 50nM RGB-286638, with or without BMSCs. The emerging role of miRNAs in the pathogenesis of multiple myeloma (MM) led us to hypothesize that the miRNA network might be among the inducible transcriptional alterations consequent to MM-bone marrow stromal cell (BMSC) interactions. Our data suggests that BMSC induced MM transcription led to aberrant miRNA expression. We therefore hypothesized that agents interfering with RNAPII transcription might inhibit aberrant miRNA expression in MM. To test this hypothesis we used RGB-286638, a novel protein kinase inhibitor, which works primarily via RNAPII inhibition followed by transcriptional arrest in MM cells. miRNA profiling of RGB-286638-exposed MM cells resulted in RNAPII arrest associated with reduced miRNA levels. RGB-286638 abrogated BMSCs-induced miRNAs, which correlated with growth arrest in MM cells. Analysis of RGB-286638-induced differentially-expressed miRNAs in MM cells, in the presence or absence of BMSCs, revealed RNAPII regulation of expression of BMSC-inducible miRNAs with established oncogenic functions in MM Our findings demonstrate the role of RNAPII in regulating miRNA network, suggesting a new rationale for using agents interfering with RNAPII transcription in the treatment of MM. TaqMan Low-Density Array (TLDA) using human miRNA version 2.0A and version 3.0B cards (Applied Biosystems) were applied to examine the global change of miRNA expression levels in MM.1S cells when co-cultured with BMSCs, with or without RGB-286638 treatment. A total of 756 mature miRNA updated in the Sanger miRBase v.15.0 were quantified according to the manufacturer's instructions as previously described. miRNAs with Ct values higher than 37 were excluded from the analysis. Normalization was carried out with the mean of RNU44 and RNU48. Relative quantification of miRNA expression was calculated with the 2M-bM-^HM-^RM-NM-^TM-NM-^TCt Ct method using the ddCt program (Shannon McCormack Advanced Molecular Diagnostics Laboratory Research Services). The data was presented as log10 of the relative quantity of each miRNA.
Project description:To gain more insight in the biological expression and role of microRNAs in trophoblast cells, we then set up to determinate the miRNA expression pattern of four trophoblastic cell lines and compare them with the miRNA signature of isolated trophoblast Total RNA was reverse-transcribed, pre-amplified with miRNA-specific primers, and loaded into TLDA cards. Quantitative real-time PCR was performed for 768 miRNAs. For each of two cards per sample (A and B), 9 ul of pre-amplified diluted sample was mixed with TaqMan reaction mix (Applied Biosystems) and loaded into miRNA TLDA cards (Applied Biosystems) by centrifugation. Cards were sealed, and qRT-PCR was performed with a real-time thermocycler (ABI-7900, Applied Biosystems) per manufacturer's recommendations.
Project description:MicroRNA (miRNA) expression profiling identified miR-638 as one of the most significantly overexpressed miRNAs in metastatic lesions compared with primary melanomas. miR-638 enhanced the tumourigenic properties of melanoma cells in vitro and lung colonization in vivo. mRNA expression profiling of miR-638 and antagomir-transduced cells identified new candidate genes as miR-638 targets, the majority of which is involved in p53-mediated apoptosis regulation. miR-638 depletion stimulated expression of p53 and its downstream target genes and induced apoptosis and autophagy in melanoma cells. miR-638 promoter analysis revealed transcription factor associated protein 2-M-NM-1 (TFAP2A) as a direct negative regulator of miR-638. Further analyses provided strong evidence for a double negative regulatory feedback loop between miR-638 and TFAP2A. Taken together, miR-638 may support melanoma progression by suppressing p53-mediated apoptosis pathways and by targeting the transcriptional repressor TFAP2A. TaqManM-BM-. low-density arrays (TLDA; human microRNA Cards A v2.1 & B v2.0, Applied Biosystems, Darmstadt, Germany) were used for measuring the expression of 667 human miRNAs in primary melanomas (PM, n=8), lymph node metastases (LNM, n=9) or distant cutaneous metastases (DCM, n=10).
Project description:Aim of this study was to identify miRNAs which are regulated by LIF in different trophoblastic cell lines Total RNA was reverse-transcribed, pre-amplified with miRNA-specific primers, and loaded into TaqMan Arrays. Quantitative real-time PCR was performed for 768 miRNAs. For each of two cards per sample (A and B), 9 ul of pre-amplified diluted sample was mixed with TaqMan reaction mix (Applied Biosystems) and loaded into miRNA TLDA cards (Applied Biosystems) by centrifugation. Cards were sealed, and qRT-PCR was performed with a real-time thermocycler (ABI-7900, Applied Biosystems) per manufacturer's recommendations.
Project description:Dynamic miRNA expression data in 377 miRNA present on Taqman low density array (TLDA) Human MicroRNA Array (A) plates (Applied Biosystems, #4398965). Primary human leukaemic B-cells were cultured for 24 hours on a stromal cell layer or a stromal cell layer with CD154 and IL-4 in order to find out how miRNA expression compared to that of freshly isolated leukaemic cells.
Project description:Signal transducer and activator of transcription 3 (STAT3) is a critical transcription factor in cancer. However, while the protein-coding target genes of STAT3 have been extensively studied, the microRNA target genes of STAT3 are less understood. MicroRNAs are short, non-coding RNAs that regulate messenger RNAs through translational inhibition and transcript degradation. They have been found to be involved in all aspects of cancer biology. Given the roles of both STAT3 and miRNAs in cancer, the function of STAT3 as a transcription factor, and the dearth of known STAT3 miRNA targets, our goal was to identify novel STAT3 miRNA targets relevant to cancer. To do so, we engineered MCF-10A cells with doxycycline-inducible expression of STAT3C. STAT3C is a constitutively-active mutant version of STAT3. Although STAT3 can be activated by various growth factors and kinases, other pathways can be activated as well, which would confound analysis of the results. Thus, the advantage of STAT3C is that it allowed specific and focused activation of STAT3 alone, and this screen represents the first genome-wide survey of miRNA expression changes associated specifically with STAT3 activity. MCF-10A, a non-transformed breast epithelial cell line, was chosen because STAT3C has been reported to be sufficient to cause their neoplastic transformation. Therefore, we reasoned that analysis of STAT3C’s effects in MCF-10A cells would be especially informative for STAT3-regulated miRNAs relevant to cancer. As a result of our study, we identified previously-known as well as novel miRNA targets of STAT3. Doxycycline-inducible MCF-10A cells were seeded, and then untreated (grown in standard growth media alone) or treated with 2?g/ml doxycycline for 48hr. Each condition was performed in biological triplicate (labeled A, B, and C), for a total of 6 samples. Total RNA was harvested and submitted to the Dana-Farber Cancer Institute Molecular Diagnostics Laboratory. MicroRNA expression profiling was performed using TaqMan Low-Density Arrays (TLDA), human miRNA version 2.0A and version 3.0B cards (Applied Biosystems).
Project description:We profiled miRNA expression using TaqMan TLDA assays to identify miRNAs with expression levels that were affected by differences in culturing conditions during monocyte-to-macrophage differentiation. 9 samples: 3 sample types for 3 different human donors
Project description:We determined expression profiles of 667 miRNAs using TaqMan Low Density Arrays (TLDA-TaqMan Array Human MicroRNA Card Set v2.0, Applied Biosystems) in 8 samples of colorectal cancer tissues and 8 samples of paired non-tumoral colonic tissues.
Project description:Eosinophlic esophagitis (EoE) is increasely recognized as an antigen-drived disorder. The goal of this study is to reveal the miRNA expression changes in EoE before and after a successful glucocorticoid steroid treatment. Total RNA was extracted from the esophageal epithelial layers of 5 paired paraffin-embedded biopsies before and after treatment with glucocorticosteroids using RecoverAll Total Nucleic Acid Extraction Kit for FFPE tissues (Ambion, Austin, TX). Five nanograms of total RNA was reverse-transcribed using the Taqman MicroRNA Reverse Transcription Kit and the Megaplex RT primer Human Pool A (Applied Biosystems). The reverse-transcribed cDNA was then pre-amplified in 12 cycles of PCR using Taqman PreAmp Master Mix and the Megaplex PreAmp primers, Human Pool A (Applied Biosystems). The cDNA’s were then diluted and loaded on to a Taqman Human miRNA Array card A (Platform GPL9731 ; Applied Biosystems), which contains probes for 377 distinct miRNAs. The Array cards were run on an ABI HT7900 qPCR instrument. Ct values were obtained for all miRNAs represented on the cards and fold changes in expression were calculated using the delta delta Ct (ddCt) method.
Project description:microRNA expression signatures can differentiate normal and breast cancer tissues and can define specific clinico-pathological phenotypes in breast tumors. In order to further evaluate the microRNA expression profile in breast cancer, we analyzed the expression of 667 microRNAs in 29 tumors and 21 adjacent normal tissues using TaqMan Low-density arrays. 130 miRNAs showed significant differential expression (adjusted P value=0.05, Fold Change=2) in breast tumors compared to the normal adjacent tissue. Importantly, the role of 43 of these microRNAs has not been previously reported in breast cancer, including several evolutionary conserved microRNA*, showing similar expression rates to that of their corresponding leading strand. The procedure begins with the retro-transcription of 70ng of total RNA with stem-loop primers to obtain a cDNA template. A pre-amplification step was included in order to increase the concentration of the original material and to detect microRNAs that are expressed at low levels. The pre-amplified product was loaded into the TaqManM-BM-. Low Density Arrays and amplification signal detection was carried out using the 7900 FAST real time thermal cycler (ABI). A total of 29 tumor and 21 normal samples (two pools: one containing five samples, other containing 12 samples, plus 4 independent normal samples) were analyzed. 23 tumors and the two normal pools were processed by triplicate, representing 82% of the total samples.