Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Identification of small non-coding RNAs in zebrafish

ABSTRACT: MicroRNAs (miRNAs) are a new class of small RNAs of approximately 22 nucleotides in length that control eukaryotic gene expression by fine tuning mRNA translation. They regulate a wide variety of biological processes, namely developmental timing, cell differentiation, cell proliferation, immune response and infection. For this reason, their identification is essential to understand eukaryotic biology. Their small size, low abundance and high instability complicated early identification; however, cloning/Sanger sequencing and new generation genome sequencing approaches overcame most technical hurdles and are being used for rapid miRNA identification in many eukaryotes. We have applied 454 DNA pyrosequencing technology to miRNA discovery in zebrafish (Danio rerio). For this, a series of cDNA libraries were prepared from small non-coding RNAs isolated at different embryonic time points and from fully developed organs. Each cDNA library was tagged with specific sequences and was sequenced using the Roche FLX genome sequencer. This approach retrieved 90% of the 192 miRNAs previously identified by cloning/Sanger sequencing and bioinformatics and 25 novel miRNAs were predicted.

ORGANISM(S): Danio rerio

PROVIDER: GSE66718 | GEO | 2016/03/31

SECONDARY ACCESSION(S): PRJNA277780

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Similar Datasets

Identification of small non-coding RNAs in zebrafish

Project description:MicroRNAs (miRNAs) are a new class of small RNAs of approximately 22 nucleotides in length that control eukaryotic gene expression by fine tuning mRNA translation. They regulate a wide variety of biological processes, namely developmental timing, cell differentiation, cell proliferation, immune response and infection. For this reason, their identification is essential to understand eukaryotic biology. Their small size, low abundance and high instability complicated early identification; however, cloning/Sanger sequencing and new generation genome sequencing approaches overcame most technical hurdles and are being used for rapid miRNA identification in many eukaryotes. We have applied 454 DNA pyrosequencing technology to miRNA discovery in zebrafish (Danio rerio). For this, a series of cDNA libraries were prepared from small non-coding RNAs isolated at different embryonic time points and from fully developed organs. Each cDNA library was tagged with specific sequences and was sequenced using the Roche FLX genome sequencer. This approach retrieved 90% of the 192 miRNAs previously identified by cloning/Sanger sequencing and bioinformatics and 25 novel miRNAs were predicted. Small RNA libraries were prepared from different zebrafish developmental stages, namely, 24 hours post-fertilization (hpf), 72 hpf, 96 hpf, 5 days post-fertilization (dpf), 45 dpf, young adult and from adult brain, eyes, gills, heart, skin and fins.

2016-03-31 | E-GEOD-66718 | biostudies-arrayexpress

High-efficiency RNA cloning enables quantitative measurement of miRNA expression by deep sequencing

Project description:MiRNA-mediated regulation depends on the stoichiometry between miRNAs and their mRNA targets. To decipher dynamic function of this complex layer, it is critical to characterize individual miRNA species within a specific cellular context. Small RNA cloning followed by deep sequencing is uniquely positioned as a genome-wide profiling method to quantify miRNA expression with potentially unlimited dynamic range and provide single-nucleotide resolution for precise miRNA classification and de novo discovery. However, significant biases introduced by RNA ligation steps in the current RNA cloning protocol often lead to inaccurate miRNA quantification by >1000-fold deviation. As a result, it has greatly hindered the broad application of this method. Here we report a highly efficient RNA cloning method that achieves over 90% efficiency for both 5’ and 3’ ligations with diverse small RNA substrates. When applied to a pool of either equimolar or differentially mixed synthetic miRNAs, the deviation of the cloning frequency for each miRNA is minimized to less than 2-fold of the anticipated value. By using samples obtained from multiple tissues and cells, we further demonstrate the accurate quantification of miRNA expression over a dynamic range of four orders of magnitude. Our results also reveal that most cistronic miRNAs are expressed at similar levels and, in each cell population, miRNAs repress their cognate targets in a dosage dependent manner. Collectively, our high-efficiency RNA cloning method combining with deep sequencing establishes a cost-effective approach for accurate genome-wide miRNA profiling. We designed an artificial system composed of synthetic miRNAs for benchmarking biases in small RNA cDNA cloning for NGS.

2013-09-18 | E-GEOD-47858 | biostudies-arrayexpress

Engineered transfer RNAs for suppression of premature termination codons

Project description:PTCs cause a multitude of human diseases and there are no established therapeutic options for their therapeutic management. Herein, we report the high-throughput cloning and identification, characterization and functional analysis of anticodon-edited tRNA which display efficacious PTC reversion in eukaryotic cells and mouse skeletal muscle. Notably, our screen identifies ACE-tRNA, in total, with the potential to repair a vast majority of known human disease-causing PTC, but this therapeutic will require overcoming tissue and delivery specific challenges. However, the engineered tRNA, once delivered, faithfully encode their cognate amino acid, thus abrogating spurious effects on downstream protein stability, folding, and trafficking, and consequently negating the need for tandem therapies involving protein folding or trafficking agents. When transfected as cDNA, ACE-tRNAs rescued multiple full-length proteins via PTC suppression; a NLuc luciferase reporter, a model protein HDH, and two disease nonsense mutations in CFTR.

2019-02-11 | PXD012581 | Pride

A unified strategy to clone modified/unmodified small RNAs and mRNAs for high throughput sequencing

Project description:The high-throughput sequencing has become a standard tool for analyzing gene expression. Usually a cDNA/DNA library is constructed with 5' and 3' linkers and then sequenced. Unlike mRNA, small RNA often contains modifications including 5' cap or triphosphate and 3' methylation, affecting the linker addition during cloning processes. Small RNA is expressed at much lower levels than mRNA, making it more difficult to clone small RNA using a small amount of total RNA. Here we have developed a new strategy to clone modified/unmodified small RNA in an all-liquid-based reaction in a single PCR tube using as little as 20 ng total RNA. The 7-hour cloning process only needs ~1 hour labor time. Moreover, this method can be used to clone mRNA, simplifying the need to prepare two cloning systems for small RNA and mRNA. Since the linkers used are derived from the Illumina Truseq linkers for DNA cloning, the PCR primers based on the linker sequences can be used to obtain amplicons derived from small RNA/mRNA/DNA. Not only is our method more convenient for cloning modified RNA than available methods, but it is also more sensitive, accurate and versatile. Moreover, the all-liquid-based reaction can be performed in an automated manner.

2020-01-24 | GSE129664 | GEO

Expression profiling data from mid-log phase cultures of wild type Vibrio campbellii BAA-1116

Project description:Small RNA and regulatory RNA discovery has been based on (i) computational predictions mainly in intergenic regions, (ii) microarray experiments, (iii) shotgun cloning of cDNA libraries, (iv) classical cloning of abundant small RNAs after size fractionation in polyacrylamide gels and (v) copurification with proteins like Hfq, CsrA and RNA polymerase. In this study, we attempted to experimentally validate in silico small and regulatory RNA predictions for V. campbellii BAA-1116 using a microarray expression profiling strategy. Specifically, we investigated transcripts from mid-log phase cultures grown in nutrient rich media and examined their hybridization to tiled probes targeting annotated V. campbellii BAA-1116 intergenic regions. Keywords: small RNA discovery

2010-06-01 | GSE20960 | GEO

Systematic and single cell analysis of Xenopus Piwi-interacting RNAs and Xiwi

Project description:Various small RNA libraries from purified microtubules or Xiwi immunoprecipitates or total extract from X.tropicalis or X.laevis egg extract. Various small RNA libraries from single X.tropicalis eggs. Analysis of this series of files is described in the manuscript "Systematic and single cell analysis of Xenopus Piwi-interacting RNAs and Xiwi". Small RNAs were ligated with linkers and converted to cDNA by reverse transcription. cDNA library was amplified by PCR and was sequenced with either the 454 Genome Sequencer FLX platform or the Illumina GA-II platform.

2009-08-31 | E-GEOD-15556 | biostudies-arrayexpress

Deep sequencing of the degradome cDNA library in soybean

Project description:We deep sequenced a degradome library constructed from different soybean tissues. As a result, 19,830,257 represented 5,337,590 distinct signatures were obtained. 70.98% of the signatures were assigned to one soybean cDNA sequence and 24.05% matched with two cDNA sequences. 428 potential targets of small RNAs and 25 novel miRNA families were identified in soybean. A total of 211 potential miRNA targets including 150 conserved miRNA targets and 69 soybean-specific miRNA targets were identified. The signatures distribution on soybean primary miRNAs (pri-miRNAs) showed that most of the pri-miRNAs had the characteristic pattern of Dicer processing. The TAS3 small RNAs (siRNAs) biogenesis was conserved in soybean and nine Auxin Response Factors (ARFs) were identified as the TAS3 siRNA targets. The global identification of miRNAs targets would contribute to the functional research of the miRNA in soybean.

2011-11-02 | GSE33379 | GEO

High-efficiency RNA cloning enables quantitative measurement of miRNA expression by deep sequencing

2013-09-18 | GSE47858 | GEO

Triticum aestivum strain:Chinese Spring

Project description:Wheat full-length cDNA sequencing by Sanger sequencer

| PRJDB4671 | ENA

Identification of miRNA from Porphyra yezoensis by high-throughput sequencing and bioinformatics analysis

Project description:This study provides a first large-scale cloning and characterization of Porphyra miRNAs and their predicted targets. These miRNAs belong to 224 conserved miRNA families and 7 are novel in Porphyra. These miRNAs add to the growing database of new miRNA and lay the foundation for further understanding of miRNA function in the regulation of Porphyra yezoensis development. We constructed a small RNA library from Porphyra yezoensis.

2010-06-04 | E-GEOD-20534 | biostudies-arrayexpress

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data