Ontology highlight
ABSTRACT:
PROVIDER: PRJNA975841 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:Transient plasmid transfection is common approach for studies in cultured mammalian cells. To examine behavior of transfected plasmids, we analyzed their transcriptional landscape by deep sequencing. We found that plasmids generate different levels of transcripts virtually everywhere. Spurious transcription may have undesirable effects as some co-transfected plasmids inhibited expression of luciferase reporters in a dose-dependent manner. In one case, we attributed this effect to kan/neo resistance cassette, which generated a unique population of edited sense and antisense small RNAs. The unexpected complexity of expression of transiently transfected plasmids highlights the importance of appropriate experimental controls.
2012-08-17 | GSE36062 | GEO
Project description:In mammals, double-stranded RNA (dsRNA) plays roles in sequence-specific RNA interference, sequence-independent interferon response, and RNA editing by adenosine deaminases. We have previously shown that long hairpin dsRNA expression in cultured cells does not activate the interferon response, it is poorly processed into siRNAs, and it is partially edited. Here, we demonstrate that dsRNA expressed from transiently transfected plasmids strongly inhibits expression of co-transfected reporter plasmids but not expression of endogenous genes or reporters stably integrated in the genome. The inhibition is concentration-dependent and independent of a cell type, transfection method, or dsRNA sequence. The inhibition occurs at the level of translation and is mediated by protein kinase R (PKR). PKR binds the expressed dsRNA, becomes phosphorylated and changes its distribution along polysome fractions. In conclusion, we demonstrate that expression from plasmids is selectively repressed if one of co transfected plasmids produces dsRNA. Our results highlight the importance of proper controls and careful interpretation of co-transfection experiments.
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Project description:We wished to assess the effect of miR196a on overall gene expression in reducing it in high expressing epithelial cells (D19 and B16) using antimiR196a and over expressing it in low-expressing cells (OKF4) using pre-miR196a. We transfected anti miR196a in to D19 and B16 oral keratinocytes which express high levels of miR196a in triplicate cultures, harvesting 24h after transfection. We transfected pre-miR196 in okf4 immorualised normal oral keratinocytes in duplicate culture. Successful transfection was ensured by qPCR. 16 Samples in total. 2 high expressing cells: control and anti-miR transfected, in triplicate. 1 low expressing cell: control and premiR transfected, in duplicate
2013-11-28 | E-GEOD-52810 | biostudies-arrayexpress
Project description:To gain insight into changes of the proteome of the rat beta cell line Ins1E with and without ablation of CerS2, shotgun proteomics was performed in control cells (transfected withCas9 plasmid without gRNA) and CerS2 KO cells (transfected with Cas9 plasmids with 2 gRNAs, ablating most of the coding exons). Ablation of CerS2 was confirmed by PCR, qPCR and Western Blot.
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Project description:Transfected double strand DNA were required for the efficient activation of STING to activate innate immune cytokine. We used microarrays to evaluate the innate immune cytokine expression in B16-OVA cells transfected with double strand DNA.
2018-09-04 | GSE110930 | GEO
Project description:Transient plasmid transfection is common approach for studies in cultured mammalian cells. To examine behavior of transfected plasmids, we analyzed their transcriptional landscape by deep sequencing. We found that plasmids generate different levels of transcripts virtually everywhere. Spurious transcription may have undesirable effects as some co-transfected plasmids inhibited expression of luciferase reporters in a dose-dependent manner. In one case, we attributed this effect to kan/neo resistance cassette, which generated a unique population of edited sense and antisense small RNAs. The unexpected complexity of expression of transiently transfected plasmids highlights the importance of appropriate experimental controls. HEK293 cells (human origin) transiently transfected with 4 various plasmids
2012-08-17 | E-GEOD-36062 | biostudies-arrayexpress
Project description:We wished to assess the effect of miR196a on overall gene expression in reducing it in high expressing epithelial cells (D19 and B16) using antimiR196a and over expressing it in low-expressing cells (OKF4) using pre-miR196a. We transfected anti miR196a in to D19 and B16 oral keratinocytes which express high levels of miR196a in triplicate cultures, harvesting 24h after transfection. We transfected pre-miR196 in okf4 immorualised normal oral keratinocytes in duplicate culture. Successful transfection was ensured by qPCR.
2013-11-28 | GSE52810 | GEO
Project description:In mammals, double-stranded RNA (dsRNA) plays roles in sequence-specific RNA interference, sequence-independent interferon response, and RNA editing by adenosine deaminases. We have previously shown that long hairpin dsRNA expression in cultured cells does not activate the interferon response, it is poorly processed into siRNAs, and it is partially edited. Here, we demonstrate that dsRNA expressed from transiently transfected plasmids strongly inhibits expression of co-transfected reporter plasmids but not expression of endogenous genes or reporters stably integrated in the genome. The inhibition is concentration-dependent and independent of a cell type, transfection method, or dsRNA sequence. The inhibition occurs at the level of translation and is mediated by protein kinase R (PKR). PKR binds the expressed dsRNA, becomes phosphorylated and changes its distribution along polysome fractions. In conclusion, we demonstrate that expression from plasmids is selectively repressed if one of co transfected plasmids produces dsRNA. Our results highlight the importance of proper controls and careful interpretation of co-transfection experiments. HEK293 cells (human origin) were transiently transfected with hairpin dsRNA-expressing (MosIR) and control (pCag) plasmids.
2013-12-27 | E-GEOD-46959 | biostudies-arrayexpress
Project description:Mouse melanoma B16 cells were transfected with either siCtrl of siTbx2 for 48 h, then RNA extracted and use in library preparation and following sequencing.
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Project description:miRNAs profiling of HepG2 cells comparing vector-control treated HepG2 cells with HepG2 cells transfected with Twist1, Bcl-2, and Twist1/Bcl-2 plasmids. Microarray analysis revealed a panel of miRNAs with significant differential expression among these four HCC cell lines.
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