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Unamplified mRNA abundance in miR-124 and mock-transfected cells

ABSTRACT: To compare total RNA levels in miR-124 and mock-transfected cells (Figure S3), 5-10 ug of total RNA from miR-124-transfected cells or mock-transfected cells or universal reference RNA (Stratagene Cat# 740000) was reverse transcribed with Superscript III (Invitrogen Cat# 18080085) in the presence of aminoallul-dUTP 5-(3-aminoallyl)-dUTP (Ambion Cat# AM8439) and natural dNTPs (GE Healthsciences Cat# US77212) with 10 ug of N9 primer (Invitrogen). Subsequently, amino-allyl-containing cDNAs from miR-124 and mock-transfected cells were covalently linked to Cy5 NHS-monoesters, and universal reference cDNA was covalently linked to Cy3 NHS-monoesters (GE HealthSciences Cat# RPN5661). Cy5- and Cy3-labeled cDNAs were mixed and diluted into 50 ul of solution containing 3x SSC, 25 mM Hepes-NaOH (pH 7.0), 20 ug human Cot-1 DNA (Invitrogen Cat# 15279011), 20 ug of poly(A) RNA (Sigma Cat# P9403), 25 ug of yeast tRNA (Invitrogen Cat# 15401029), and 0.3% SDS. The sample was incubated at 95 C for 2 min, spun at 14,000 rpm for 10 mins in a microcentrifuge, then hybridized at 65 C An all pairs experiment design type is where all labeled extracts are compared to every other labeled extract.

ORGANISM(S): Homo sapiens  

SUBMITTER: Daniel Hogan   Patrick O Brown 

PROVIDER: E-GEOD-18837 | ArrayExpress | 2010-06-20



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Concordant regulation of translation and mRNA abundance for hundreds of targets of a human microRNA.

Hendrickson David G DG   Hogan Daniel J DJ   McCullough Heather L HL   Myers Jason W JW   Herschlag Daniel D   Ferrell James E JE   Brown Patrick O PO  

PLoS biology 20091110 11

MicroRNAs (miRNAs) regulate gene expression posttranscriptionally by interfering with a target mRNA's translation, stability, or both. We sought to dissect the respective contributions of translational inhibition and mRNA decay to microRNA regulation. We identified direct targets of a specific miRNA, miR-124, by virtue of their association with Argonaute proteins, core components of miRNA effector complexes, in response to miR-124 transfection in human tissue culture cells. In parallel, we asses  ...[more]

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