Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Small RNA profiling in human cell line following Dicer silencing

ABSTRACT: microRNAs (miRNAs) are a large class of small non-coding RNAs which post-transcriptionally regulate the expression of a large fraction of all animal genes and are important in a wide range of biological processes. Recent advances in high-throughput sequencing allow miRNA detection at unprecedented sensitivity, but the computational task of accurately identifying the miRNAs in the background of sequenced RNAs remains challenging. For this purpose we have designed miRDeep2, a substantially improved algorithm which identifies canonical and non-canonical miRNAs such as those derived from transposable elements and informs on high-confidence candidates that are detected in multiple independent samples. Analyzing data from seven animal species representing the major animal clades, miRDeep2 identified miRNAs with an accuracy of 98.6-99.9% and reported hundreds of novel miRNAs. To test the accuracy of miRDeep2, we knocked down the miRNA biogenesis pathway in a human cell line and sequenced small RNAs before and after. The vast majority of the >100 novel miRNAs expressed in this cell line were indeed specifically down-regulated, validating most miRDeep2 predictions. Last, a new miRNA expression profiling routine, low time and memory usage and user-friendly interactive graphic output can make miRDeep2 useful to a wide range of researchers." high-throughput sequencing was used to profile small RNA expression in a human MCF-7 cell line before and after Dicer knock-down

ORGANISM(S): Homo sapiens

SUBMITTER: Marc Friedlaender

PROVIDER: E-GEOD-31069 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

miRDeep2 accurately identifies known and hundreds of novel microRNA genes in seven animal clades.

Friedländer Marc R MR Mackowiak Sebastian D SD Li Na N Chen Wei W Rajewsky Nikolaus N

Nucleic acids research 20110912 1

microRNAs (miRNAs) are a large class of small non-coding RNAs which post-transcriptionally regulate the expression of a large fraction of all animal genes and are important in a wide range of biological processes. Recent advances in high-throughput sequencing allow miRNA detection at unprecedented sensitivity, but the computational task of accurately identifying the miRNAs in the background of sequenced RNAs remains challenging. For this purpose, we have designed miRDeep2, a substantially improv ...[more]

PMID: 21911355

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Project description:MicroRNAs (miRNAs) play a important part in post-transcriptional gene regulation and have been shown to control many genes involved in various biological and metabolic processes. There have been extensive studies to discover miRNAs and analyze their functions in model plant species, such as Arabidopsis and rice and other plants. However, the number of miRNAs discovered in grape is relatively low and little is known about miRNAs responded gibberellin during fruit germination. In this study, a small RNA library from gibberellin grape fruits was sequenced by the high throughput sequencing technology. A total of 16,033,273 reads were obtained. 812,099 total reads representing 1726 unique sRNAs matched to known grape miRNAs. Further analysis confirmed a total of 149 conserved grapevine miRNA (Vv-miRNA) belonging to 27 Vv-miRNA families were validated, and 74 novel potential grapevine-specific miRNAs and 23 corresponding novel miRNAs* were discovered. Twenty-seven (36.5%) of the novel miRNAs exhibited differential QRT-PCR expression profiles in different development gibberellin-treated grapevine berries that could further confirm their existence in grapevine. QRT-PCR analysis on transcript abundance of 27 conserved miRNA family and the new candidate miRNAs revealed that most of them were differentially regulated by the gibberellin, with most conserved miRNA family and 26 miRNAs being specifically induced by gibberellin exposure. All novel sequences had not been earlier described in other plant species. In addition, 117 target genes for 29 novel miRNAs were successfully predicted. Our results indicated that miRNA-mediated gene expression regulation is present in gibberellin-treated grape berries. This study led to the confirmation of 101 known miRNAs and the discovery of 74 novel miRNAs in grapevine. Identification of miRNAs resulted in significant enrichment of the gibberellin of grapevine miRNAs and provided insights into miRNA regulation of genes expressed in grape berries. GSM604831 is the control for the gibberellin-treated sample. The mixture samples of young berries (one week after flowering) large berries (five week after flowering after flowering), and old berries (nine week after flowering) treated with gibberellin, respectively, were generated by deep sequencing, in triplicate, using Illumina 1G Genome Analyzer.

Project description:Small RNAs (sRNAs) are emerging as important regulators of biological processes in plants. To characterize the small RNA species and expression changes in ‘Anliu’ sweet orange (wild type, WT) and its red-flesh mutant (MT) with lycopene accumulation, we used high-throughput pyrosequencing to identify and quantitatively profile sRNAs. We identified 112 known miRNAs belonging to 64 families from the sweet orange. Comparative analysis revealed that 63 of the 112 known miRNAs exhibited significant expression differences between WT and MT. We also identified 12 novel miRNAs and 9 miRNA candidates from sweet orange; the 12 novel miRNAs are in line with the biogenesis characteristics including the stem-loop structure of the pre-miRNAs and existence of the miRNA*s in the sRNA libraries. Comparative profiling revealed that 10 novel miRNAs and 8 miRNA candidates are differentially expressed between WT and MT. Potential targets of these differentially expressed miRNAs included several important genes that are involved in carotenoid biosynthesis, such as geranylgeranyl pyrophosphate synthase (GGPS) and lycopene β-cyclase (LYCb). Moreover, GO and KEGG annotation revealed that high ranked miRNA-target genes are those implicated in transcription regulation, protein modification and photosynthesis. We proposed that miRNA-target genes are the hot-spots for generating sRNAs. This study provides the first large scale cloning and characterization of citrus miRNAs, which also lays a foundation for unraveling the mechanism of lycopene accumulation in the sweet orange mutant on post-transcription level. Size fractionated small RNAs (16-30 bp) from total RNA extracts was ligated to 5' and 3' adapters, and reverse transcribed. After PCR amplification the sample was subjected to Solexa sequencing. The resultant 35nt sequence data were filtered according to base quality value. The remained sequences were used to trim 5' and 3' adaptors. The clean tags were used for further analysis.

Dataset Information

Small RNA profiling in human cell line following Dicer silencing

Publications

miRDeep2 accurately identifies known and hundreds of novel microRNA genes in seven animal clades.

Similar Datasets

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