Transcription profiling by array of human pancreatic cancer cell line to investigate off-target effects of RAD17 RNAi by shRNA transduction
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ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Fredebohm J
PROVIDER: S-ECPF-MTAB-1535 | biostudies-other | 2013 Aug
REPOSITORIES: biostudies-other
Similar Datasets
Project description:To examine off-target effects of RAD17 RNAi by shRNA transduction, expression profilling was performed.
2013-06-03 | E-MTAB-1535 | biostudies-arrayexpress
Project description:BACKGROUND: The identification of novel drug targets by assessing gene functions is most conveniently achieved by high-throughput loss-of-function RNA interference screening. There is a growing need to employ primary cells in such screenings, since they reflect the physiological situation more closely than transformed cell lines do. Highly miniaturized and parallelized approaches as exemplified by reverse transfection or transduction arrays meet these requirements, hence we verified the applicability of an adenoviral microarray for the elucidation of gene functions in primary cells. RESULTS: Here, we present microarrays of infectious adenoviruses encoding short hairpin RNA (shRNA) as a new tool for gene function analysis. As an example to demonstrate its application, we chose shRNAs directed against seven selected human protein kinases, and we have performed quantitative analysis of phenotypical responses in primary human umbilical vein cells (HUVEC). These microarrays enabled us to infect the target cells in a parallelized and miniaturized procedure without significant cross-contamination: Viruses were reversibly immobilized in spots in such a way that the seeded cells were confined to the area of the viral spots, thus simplifying the subsequent addressing of genetically modified cells for analysis. Computer-assisted image analysis of fluorescence images was applied to analyze the cellular response after shRNA expression. Both the expression level of knock-down target proteins as well as the functional output as measured by caspase 3 activity and DNA fractionation (TUNEL) were quantified. CONCLUSION: We have developed an adenoviral microarray technique suitable for miniaturized and parallelized analysis of gene function. The practicability of this technique was demonstrated by the analysis of several kinases involved in the activation of programmed cell death, both in tumor cells and in primary cells.
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Project description:ER-dependent gene expression was investigated in the LY2 endocrine resistant cell line by treatment with ICI 182780. Cells were steroid depleted for 3 days prior to treatment with ICI for 6 hours. Four biological replicates were processed and analysed.
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Project description:RNA interference (RNAi) is a powerful tool for studying gene function owing to the ease with which it can selectively silence genes of interest, and it has also attracted attention because of its potential for therapeutic applications. Chemically synthesized small interfering RNAs (siRNAs) and DNA vector-based short hairpin RNAs (shRNAs) are now widely used as RNAi triggers. In contrast to expressed shRNAs, the use of synthetic shRNAs is limited. Here we designed shRNAs modeled on a precursor microRNA (pre-miRNA) and evaluated their biological activity. We demonstrated that chemically synthetic pre-miRNA-based shRNAs have more potent RNAi activity than their corresponding siRNAs and found that their antisense strands are more efficiently incorporated into the RNA-induced silencing complex. Although greater off-target effects and interferon responses were induced by shRNAs than by their corresponding siRNAs, these effects could be overcome by simply using a lower concentration or by optimizing and chemically modifying shRNAs similar to synthetic siRNAs. These are challenges for the future.
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Project description:Lentiviral vectors have become mainstream gene transfer vehicles for their ability to deliver and integrate into host cells. In RNA interference (RNAi) applications, lentiviral constructs constitutively express double-stranded RNA molecules, usually as short hairpin RNA (shRNA), enabling long-term gene silencing, and, when pseudotyped with a broad host glycoprotein envelope, allow a multitude of cell types to be transduced. Their successful use ultimately relies on the production of lentiviral particles in high titer and uniformity. Typical methods require the transfection of three or more plasmids in which essential viral elements have been encoded separated so as to remain replication deficient. These transfection procedures are of critical importance; however, methods often vary among laboratories, making it difficult to assess the overall efficiency of lentiviral particle production. In this report, we focus exclusively on this step and compare the overall impact of the commercial transfection reagent FuGENE 6 with FuGENE HD. We found that FuGENE HD resulted in at least 5-fold improvement in viral particle titer as assessed by the p24 standard enzyme-linked immunosorbent assay. We present the complete optimized workflow and demonstrate this utility in which a single modification of this transfection step improved the lentiviral particle production.
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Project description:RNAi gene therapies for HIV-1 will likely need to employ multiple shRNAs to counter resistant strains. We evaluated 3 shRNA co-expression methods to determine their suitability for present use; multiple expression vectors, multiple expression cassettes and single transcripts comprised of several dsRNA units (aka domains) with each being designed to a different target. Though the multiple vector strategy was effective with 2 shRNAs, the increasing number of vectors required is a major shortcoming. With single transcript configurations we only saw adequate activity from 1 of 10 variants tested, the variants being comprised of 2 - 3 different target domains. Whilst single transcript configurations have the most advantages on paper, these configurations can not yet be rapidly and reliably re-configured for new targets. However, our multiple cassette combinations of 2, 3 and 4 (29 bp) shRNAs were all successful, with suitable activity maintained in all positions and net activities comparable to that of the corresponding single shRNAs. We conclude that the multiple cassette strategy is the most suitably developed for present use as it is easy to design, assemble, is directly compatible with pre-existing shRNA and can be easily expanded.
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Project description:Existing transgenic RNAi resources in Drosophila melanogaster based on long double-stranded hairpin RNAs are powerful tools for functional studies, but they are ineffective in gene knockdown during oogenesis, an important model system for the study of many biological questions. We show that shRNAs, modeled on an endogenous microRNA, are extremely effective at silencing gene expression during oogenesis. We also describe our progress toward building a genome-wide shRNA resource.
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Project description:We used microarrays to identify the expression differences of FKBP5 gene between the pancreatic tumor and normal samples. On average normal samples had more FKBP5 expression compared to tumor samples .Experiment Overall Design: This experiment consists of 36 tumor samples and 16 normal samples; a total of 52 samples. 16 samples consist of both tumor and normal expression data, whereas 20 samples consist of only tumor data.
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Project description:siRNA-mediated inhibition compared to untreated cells and cells transfected with nonsense siRNA. Loss of contact inhibition and anchorage-independent growth are hallmarks of cancer cells. In this context, frequent inactivation of the Hippo pathway and subsequent nuclear enrichment of the transcriptional coactivator yes-associated protein (YAP) uncouple cell proliferation and anti-apoptosis from contact inhibition, associated with uncontrolled tumor growth and tumor cell dissemination. However, general molecular mechanisms of tumor-supporting YAP activity remain unclear. In this study, we show that overexpression and nuclear accumulation of YAP in hepatocytes and hepatocellular carcinoma (HCC) cells leads to an induction of the Notch pathways through transcriptional activation of the Notch ligand jagged-1 (Jag-1). This induction of Jag-1 strictly depends on binding of YAP to TEAD4 and does not rely on WNT/β-catenin pathway activity. Co-activation of YAP, TEAD4, Jag-1, and the Notch target gene Hes-1 was significantly higher in HCC from patients with poor prognosis. High-level expression and nuclear accumulation of YAP correlates with Jag-1/Notch activation not only in human HCC tissues, but also in colon and pancreatic cancer tissues. Thus, our data demonstrate that YAP-driven co-activation of the Jag-1/Notch pathway in part facilitates oncogenic properties of the oncogene YAP not only in HCC but also in different gastrointestinal malignancies. Expression profiling of untreated HCC cell lines (control 1), cells transfected with scrambled/nonsense siRNA (control 2), and after siRNA-mediated YAP inhibition.
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Project description:Expression profiling of a panel of metastatic melanoma cell lines
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