Project description:This SuperSeries is composed of the following subset Series: GSE11079: mRNAs associated with Ago2: Ago2, Ago2+miR-1, Ago2+miR-124, mock transfected GSE11080: Expression data HEK293T cells transfected with either Ago2, Ago2 + miR-1, Ago2 + miR-124, mock GSE11081: miRNA arrays: Ago2 transfected vs. Mock, Ago2 IP vs. total RNA Keywords: SuperSeries Refer to individual Series
Project description:miRNAs were enriched from HEK293T cells using the ambion FLASHPAGE fractionater after mock transfection, Ago2 transfection, and FLAG-Ago2 IP. miRNAs were labeled and hybridized: Ago2 transfected vs. mock transfection and Ago2 transfected vs. Ago2 IPs. Set of arrays that are part of repeated experiments Keywords: Biological Replicate
Project description:miRNAs were enriched from HEK293T cells using the ambion FLASHPAGE fractionater after mock transfection, Ago2 transfection, and FLAG-Ago2 IP. miRNAs were labeled and hybridized: Ago2 transfected vs. mock transfection and Ago2 transfected vs. Ago2 IPs. Set of arrays that are part of repeated experiments Keywords: Biological Replicate Biological Replicate Computed
Project description:The aim of the study is to identify miRNA specific targets in Hodgkin lymphoma cell lines. By immunoprecipitation (IP) of wild type Ago2, Ago2 associated gene transcripts (ie miRNA targets) are coimmunoprecipitated. In cells transfected with anti-miR-17/20/93/106, the miR-17 seed family specific targets are not coimmunoprecipitated with Ago2. With microarray analysis, signal intensities of probes associated with Ago2 from untransfected and anti-miRNA transfected cells are compared. Gene transcripts that are depleted from the Ago2-IP fraction upon miRNA inhibition (ie miRNA specific targets) are identified.
Project description:As a core RISC component, Ago2 associates with miRNAs and target mRNAs. To identify these mRNAs, we ran lysate from HEK293T cells over a FLAG resin from 2 conditions: +FLAG-Ago2, +mock transfection. To identify mRNAs associated with specific miRNAs, we ran lysate from HEK293T cells over a FLAG resin from 2 conditions: +FLAG-Ago2 & miR-1, and +FLAG-Ago2 & miR-124. Set of arrays that are part of repeated experiments Compound Based Treatment: mock transfected Keywords: Biological Replicate
Project description:RNAs that are enriched in AGO2 Immunoprecipitated (IP) products or PIWIL1 IP products were identified from mouse(BALB/C) adult testes by examine the ratio of total RNA signal intensity to AGO2 IP RNA or PIWIL1 IP RNA signal intensity. Two-condition experiment,Total RNA extracted from mouse adult testes vs. AGO2 IP RNA extracted from mouse adult testes and total RNA extracted from mouse adult testes vs. PIWIL1 IP RNA extracted from mouse adult testes.
Project description:Transgenic Arabidopsis plants (AGO2::HA:AGO2) were treated with either mock (10 mM MgCl2) or Pseudomonas syringae pv. tomato (Pst) expressing avrRpt2 (R2) at a concentration of 2 x 107 cfu/ml for 14 hours. sRNAs associated with AGO1 and AGO2 were co-immunoprecipitated using antibodies against either AGO1 (AGO1-IP), or HA (hemagglutinin) (AGO2-IP). As controls, we also gel-purified the 18-28 nt fraction of the total RNAs from an AGO2 mutant (ago2-1). The co-immunoprecipitated or gel-purified RNAs were cloned and sequenced by Illumina deep sequencing.
Project description:Ago2 binds mature microRNAs (miRNAs) to regulate gene expression. A mutant Ago2 is constructed to render it unable to bind miRNAs and inhibit mRNA translation. Human 293T cells were transiently transfected with a plasmid that encoded a FLAG-tagged wildtype human Ago2 or mutant Ago2. FLAG IP was preformed, and assoicated RNA was isolated and subjected miRNA microarray analysis.
Project description:Mouse Embryonic Stem (ES) cells express a unique set of microRNAs (miRNAs), the miR-290-295 cluster. To elucidate the role of these miRNAs and how they integrate into the ES cell regulatory network requires identification of their direct regulatory targets. The difficulty, however, arises from the limited complementarity of metazoan miRNAs to their targets, with the interaction requiring as few as six nucleotides of the miRNA seed sequence. To identify miR-294 targets, we used Dicer1-null ES cells, which lack all endogenous mature miRNAs, and introduced just miR-294 into these ES cells. We then employed two approaches to discover miR-294 targets in mouse ES cells: transcriptome profiling using microarrays, and a biochemical approach to isolate mRNA targets associated with the Argonaute2 (Ago2) protein of the RISC (RNA Induced Silencing Complex) effector, followed by RNA-Sequencing. In the absence of Dicer1, the RISC complexes are largely devoid of mature miRNAs, and should therefore contain only transfected miR-294 and its base-paired targets. Our data suggest that miR-294 may promote pluripotency by regulating a subset of c-Myc target genes, and upregulating pluripotency-associated genes such as Lin28. For RNA-IP experiments, cells were transfected with 300 pmol of miR-294 or cel-239b control miRNA (Dharmacon) and harvested 12-16 hr after transfection for immunoprecipitation. Following RNA-immunoprecipitation of Ago2-myc, RNA from the INPUT (total RNA) and IP were subjected to library preparation and sequenced by SOLiD. There are 10 samples from Dicer1-null mouse ES cells, which are essentially 5 sample pairs of INPUT (A) and its corresponding RNA-IP (B): Ago2-myc transfected with miR-294 (sampe 1), 2 replicates of Ago2-myc-MUT (catalytically inactive) transfected with miR-294 (sample 2 & 5), 2 replicates of Ago2-myc transfected with cel-239b (sample 3 & 4).