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Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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ShRNA profiling of human cells containing varying concentrations of shRNA expression constructs to determine shRNA abundance using half hairpin sequences

ABSTRACT: RNAi screens via pooled short hairpin RNAs (shRNAs) have recently become a powerful tool for the identification of essential genes in mammalian cells. We synthesized DNA microarrays with six overlapping 25 nt long tiling probes complementary to each unique 60 nt molecular barcode sequence associated with every shRNA expression construct. By analyzing dilution series of expression constructs we show how our approach allows quantification of shRNA abundance from a pool and how it clearly outperforms the commonly used analysis via the shRNAs half hairpin sequences. In this part of the study one equimolar reference pool and three test pools of varying template concentrations were PCR amplified and hybridized to individual arrays.

ORGANISM(S): Homo sapiens

SUBMITTER: Michael Boettcher

PROVIDER: E-MEXP-2475 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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shRNA profiling of human cells containing varying concentrations of shRNA expression constructs to determine shRNA abundance using barcodes

Project description:RNAi screens via pooled short hairpin RNAs (shRNAs) have recently become a powerful tool for the identification of essential genes in mammalian cells. We synthesized DNA microarrays with six overlapping 25 nt long tiling probes complementary to each unique 60 nt molecular barcode sequence associated with every shRNA expression construct. By analyzing dilution series of expression constructs we show how our approach allows quantification of shRNA abundance from a pool and how it clearly outperforms the commonly used analysis via the shRNAs half hairpin sequences. In this part of the study one equimolar reference pool and three test pools of varying template concentrations were PCR amplified and hybridized to individual arrays.

2009-12-08 | E-MEXP-2474 | biostudies-arrayexpress

shRNA profiling of human breast carcinoma cells expressing over 300 shRNA constructs using barcode tiling arrays - screen 1

Project description:RNAi screens via pooled short hairpin RNAs (shRNAs) have recently become a powerful tool for the identification of essential genes in mammalian cells. We synthesized DNA microarrays with six overlapping 25 nt long tiling probes complementary to each unique 60 nt molecular barcode sequence associated with every shRNA expression construct.. In this part of the study pooled shRNAs were transduced into MDA-MB-231 breast carcinoma cell lines and their inhibitory effects four weeks post transduction were analyzed via microarray hybridization.

2009-12-08 | E-MEXP-2476 | biostudies-arrayexpress

shRNA profiling of human breast carcinoma cells expressing over 300 shRNA constructs using barcode tiling arrays - screens 2 and 3

Project description:RNAi screens via pooled short hairpin RNAs (shRNAs) have recently become a powerful tool for the identification of essential genes in mammalian cells. We synthesized DNA microarrays with six overlapping 25 nt long tiling probes complementary to each unique 60 nt molecular barcode sequence associated with every shRNA expression construct.. In this part of the study pooled shRNAs were transduced into MDA-MB-231 breast carcinoma cell lines and their inhibitory effects four weeks post transduction were analyzed via microarray hybridization.

2009-12-08 | E-MEXP-2477 | biostudies-arrayexpress

Methylation profiling by array of human carcinoma cells

Project description:Genomic DNA from cell lines was extracted and treated with sodium bisulfite. Fragments were PCR-amplified from the treated template DNA, labelled and hybridized to the microarray. The array contained probe sequences complementary to formerly methylated (M) and unmethylated (U) cytosines respectively. Hence the signal intensity ratio from M/(M+U) represents each cytosines methylation level in percent.

2010-05-11 | E-MEXP-2698 | biostudies-arrayexpress

Transcription profiling of pea seed development

Project description:SnRK1 (sucrose-non-fermenting-1-related) protein kinases are involved in the regulation of plant metabolism controlling both gene expression and phosphorylation. The aim of the study was to investigate the role of SnRK1 in pea seed development. To study the effect of SnRK1 deficiency, transgenic pea plants were generated carrying a gene for VfSnRK1 in antisense orientation under control of seed specific vicilin promoter. Selected transgenic lines were characterized with decreased levels of PsSnRK1 mRNA and reduced up to 71% phosphorylation activity. Antisense inhibition of SnRK1 resulted in reduced seeds fresh weight, defect of pollen development. To dissect the SnRK1-antisense phenotype at the molecular level, a search for genes with differential expression patterns in transgenic plant versus wild type seeds has been performed using cDNA macroarray analysis. Radioactive labeled cDNA probes were prepared from RNA isolated from embryo of developing seeds of wild type (11, 13, 15, 17, 19, and 21 DAP) and transgenic SnRK1-antisense plant (13, 15, 17 and 19 DAP), which correspond to the transition phase of seed development, and hybridized to cDNA macroarrays.

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Transcription profiling of Vicia narbonensis (legume) and Pisum sativum (pea) expressing a bacterial phosphoenol pyruvate carboxylase during seed maturation

Project description:In order to change assimilate partitioning during seed maturation a bacterial phosphoenolpyruvate carboxylase (PEPC) has been expressed in V. narbonensis under control of the LegB4 promoter. Transgenic bean seeds accumulate app. 20% more protein and have higher seed weight. Physiological and biochemical analyses indicate a shift of metabolic fluxes from sugars/starch to organic acids/free amino acids suggesting increased anaplerotic carbon flow. This indicates that the availability of carbon acceptors limits seed protein synthesis. Gene expression was analysed in growing embryos using macro-arrays containing 5,548 seed-specific pea genes. Radioactive labeled cDNA probes were prepared from RNA isolated from embryo of developing seeds of wild type and transgenic PEPC plants of V. narbonensis (15, 17, 19, 21, 25, and 30 DAP) and hybridized to cDNA macroarrays.

2007-11-14 | E-IPKG-2 | biostudies-arrayexpress

A genome-wide screen identifies silencers with distinct chromatin properties and mechanisms of repression

Project description:Differential gene transcription enables development and homeostasis in all animals and is regulated by two major classes of distal cis-regulatory DNA elements (CREs), enhancers and silencers. While enhancers have been thoroughly characterized, the properties and mechansisms of silencers remain largely unknown. By an unbiased genome-wide functional screen in Drosophila melanogaster S2 cells, we discover a class of silencers that bind one of three transcription factors (TFs) and are generally not included in chromatin-defined CRE catalogs, as they mostly lack detectable DNA accessibility. The silencer-binding TF CG11247, which we term Saft, safeguards cell fate decisions in vivo and functions via a highly-conserved domain we term ZAC and the corepressor G9a, independently of G9a’s H3K9-methyltransferase activity. Overall, our identification of silencers with unexpected properties and mechanisms has important implications for the understanding and future study of repressive CREs, as well as the functional annotation of animal genomes.

2024-11-13 | PXD048161 | Pride

Transcription profiling of mouse primary neural stem cells (neurospheres) originating from separate isolations (individuals), different passages and parallel cultures

Project description:Investigation of transcriptome variability in neurospheres originating from separate isolations (individuals), different passages and parallel cultures. As a control neurospheres were also compared to neurospheres induced to differentiate by adding serum and plating onto solid support.

2005-03-31 | E-MEXP-297 | biostudies-arrayexpress

Transcription profiling of mouse adult primary neural stem cells when stimulated with the proliferative agent PACAP (pituitary adenylate cyclase-activating polypeptide)

Project description:Investigation of transcriptional changes induced in adult primary neural stem cells when stimulated with the proliferative agent PACAP (pituitary adenylate cyclase-activating polypeptide). In addition the data was compared to results obtained from a proliferation control (stimulation with a transmembrane receptor agonist) and a differentiation control (primary neural stem cells induced to differentiate by withdrawing growth factors, adding serum and plating onto solid support).

2005-05-15 | E-MEXP-322 | biostudies-arrayexpress

BIGenomics_Agilent_15k_templates

Project description:Hybridization of ligation probes to a zipcode (tag) microarray. The purpose of the experiment was to establish functionality and target specificity of the ligation probes by using pools of synthetic templates with all of the probes. The templates are 80-mer synthetic dsDNA molecules, each specific for a single probe. Each probe is a ssDNA molecule containing target recognition sequences, PCR primer binding sequences and a unique tag sequence. If a matching target sequence is present in the mixture, the probes is ligated into a circular molecule, which can be PCR amplified and hybrized on a microarray harbouring complementary tag sequences.

2012-02-24 | E-MEXP-3538 | biostudies-arrayexpress

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