Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Adult Caenorhabditis elegans: wild-type (N2) and mir-243 mutant worms

ABSTRACT: Transcriptional profiling of N2 vs. mir-243 worms, aiming to identify direct and indirect targets of the microRNA. N2 and mir-243 young-adult worms grown at 20C were analyzed. Three biological replicates with dye-swaps were performed.

ORGANISM(S): Caenorhabditis elegans

SUBMITTER: Rene Ketting

PROVIDER: E-GEOD-20558 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

MicroRNA-directed siRNA biogenesis in Caenorhabditis elegans.

Corrêa Régis L RL Steiner Florian A FA Berezikov Eugene E Ketting René F RF

PLoS genetics 20100408 4

RNA interference (RNAi) is a post-transcriptional silencing process, triggered by double-stranded RNA (dsRNA), leading to the destabilization of homologous mRNAs. A distinction has been made between endogenous RNAi-related pathways and the exogenous RNAi pathway, the latter being essential for the experimental use of RNAi. Previous studies have shown that, in Caenorhabditis elegans, a complex containing the enzymes Dicer and the Argonaute RDE-1 process dsRNA. Dicer is responsible for cleaving ds ...[more]

PMID: 20386745

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Project description:Potential miR-202 targets were identified using a targetome-wide RIP-based microarray. HeLa cells were transfected with either a miR-202 mimic or a scrambled single-stranded RNA negative control. RNA was isolated from total cell lysate prior to IP and from antibody-immobilized Protein G agarose beads-RNP complexes (post-IP). Ribonucleoprotein IP was performed using the RIP-Assay kit for microRNA (MBL) according to the protocol described by the manufacturer. Briefly, anti-EIF2C2/Ago2 monoclonal antibody (Novus Biologicals, LLC) was incubated with Protein G plus agarose beads (Pierce) at 4M-BM-0C overnight to prepare antibody-immobilized beads. 20 million cells were harvested and washed four times with ice-cold DEPC-treated PBS. The cell pellet was lysed with 500 M-NM-<l of lysis buffer and the supernatant was incubated with Protein G agarose beads without antibody to reduce nonspecific adsorption. The cell lysate was then transferred into a tube containing antibody-immobilized Protein G agarose beads and incubated for 3 hours at 4M-BM-0C. Genome-wide expression levels were analyzed in total RNA samples from total cell lysate and antibody-immobilized Protein G agarose beads-RNP complexes from cells transfected with miR-202 mimic or negative control using the Agilent 44K 60-mer human whole-genome microarray. Signal hybridization and scans were performed by MOGene, LC (St Louis, MO) (run in biological duplicate). The normalized signal intensities of probes from RNP-bead complexes (post-IP fraction) were divided by the signal intensities from total cell lysate (pre-IP fraction) in miR-202 mimic-transfected cells. Transcripts were identified as members of the global miRNA targetome if they exhibited an enrichment fold change > 2.0. From this list, post-IP to pre-IP signal intensity ratios in miR-202 mimic-transfected cells were divided by post-IP to pre-IP signal intensity ratios in NC cells. Transcripts exhibiting normalized signal ratios of > 1.5 were considered to be bound with miR-202 in the RNA-induced silencing complex (RISC), and therefore potential direct miR-202 targets.

Dataset Information

Adult Caenorhabditis elegans: wild-type (N2) and mir-243 mutant worms

Publications

MicroRNA-directed siRNA biogenesis in Caenorhabditis elegans.

Similar Datasets

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