Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Screening target specificity of siRNAs by rapid amplification of cDNA ends (RACE) for non-sequenced species.

ABSTRACT: RNA interference (RNAi) is the process of sequence-specific posttranslational gene silencing triggered by double-stranded RNAs (dsRNAs). RNAi is a widely used approach for studying gene function. However, studies have shown that using siRNA can lead to off-target effects when the siRNA contains sufficient sequence identity to non-target mRNA sequences. One of the important steps in designing dsRNA is verification that it has sequence identity to only the target mRNA. In this report, we propose an approach for primary screening dsRNAs for potential off-target effects by using rapid amplification of cDNA ends. This method can be especially useful for model systems using species that have limited availability of sequence data.

SUBMITTER: Sabirzhanov B

PROVIDER: S-EPMC3992891 | biostudies-literature | 2011 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Screening target specificity of siRNAs by rapid amplification of cDNA ends (RACE) for non-sequenced species.

Sabirzhanov Boris B Sabirzhanova Inna B IB Keifer Joyce J

Journal of molecular neuroscience : MN 20110325 1

RNA interference (RNAi) is the process of sequence-specific posttranslational gene silencing triggered by double-stranded RNAs (dsRNAs). RNAi is a widely used approach for studying gene function. However, studies have shown that using siRNA can lead to off-target effects when the siRNA contains sufficient sequence identity to non-target mRNA sequences. One of the important steps in designing dsRNA is verification that it has sequence identity to only the target mRNA. In this report, we propose a ...[more]

PMID: 21437668

Similar Datasets

5' RACE (Rapid amplification of cDNA ends) and 3' RACE of U6 or U1-driven Cas13 CRISPR RNAs (crRNAs)

Project description:Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) technologies have evolved rapidly over the past decade with the continuous discovery of new Cas systems. In particular, RNA-targeting CRISPR-Cas13 proteins are promising single-effector systems to regulate target mRNAs without altering genomic DNA, yet the current Cas13 systems are still restrained by suboptimal efficiencies. Here, we show that U1-driven CRISPR RNAs (crRNAs) can dramatically increase the efficiency of various applications, including RNA knockdown and editing, without modifying the Cas13 protein effectors. We confirm that U1-driven crRNAs are exported into the cytoplasm, while conventional U6 promoter-driven crRNAs are mostly confined in the nucleus. Furthermore, we reveal that the end positions of crRNAs expressed by the U1 promoter are consistent regardless of different guide sequences and lengths. We also demonstrate that U1-driven crRNAs, but not U6-driven crRNAs, can efficiently repress the translation of target genes in combination with catalytically inactive Cas13 proteins. Finally, we show that U1-driven crRNAs can counteract the inhibitory effect of miRNAs. Our simple and effective engineering enables unprecedented cytosolic RNA-targeting applications.

2024-09-15 | GSE275245 | GEO

Dissecting efficiency of a 5' rapid amplification of cDNA ends (5'-RACE) approach for profiling T-cell receptor beta repertoire.

Project description:Deep sequencing of T-cell receptor (TCR) genes is powerful at profiling immune repertoire. To prepare a TCR sequencing library, multiplex polymerase chain reaction (mPCR) is widely applied and is highly efficient. That is, most mPCR products contain the region critical for antigen recognition, which also indicates regular V(D)J recombination. Multiplex PCR, however, may suffer from primer bias. A promising alternative is 5'-RACE, which avoids primer bias by applying only one primer pair. In 5'-RACE data, however, non-regular V(D)J recombination (e.g., TCR sequences without a V gene segment) has been observed and the frequency varies (30-80%) between studies. This suggests that the cause of or how to reduce non-regular TCR sequences is not yet well known by the science community. Although it is possible to speculate the cause by comparing the 5'-RACE protocols, careful experimental confirmation is needed and such a systematic study is still not available. Here, we examined the 5'-RACE protocol of a commercial kit and demonstrated how a modification increased the fraction of regular TCR-β sequences to >85%. We also found a strong linear correlation between the fraction of short DNA fragments and the percentage of non-regular TCR-β sequences, indicating that the presence of short DNA fragments in the library was the main cause of non-regular TCR-β sequences. Therefore, thorough removal of short DNA fragments from a 5'-RACE library is the key to high data efficiency. We highly recommend conducting a fragment length analysis before sequencing, and the fraction of short DNA fragments can be used to estimate the percentage of non-regular TCR sequences. As deep sequencing of TCR genes is still relatively expensive, good quality control should be valuable.

| S-EPMC7377388 | biostudies-literature

Adapting 3' Rapid Amplification of CDNA Ends to Map Transcripts in Cancer.

Project description:Maturation of eukaryotic mRNAs involves 3' end formation, which involves the addition of a poly(A) tail. In order to map the 3' end of a gene, the traditional method of choice is 3' rapid amplification of cDNA ends (3' RACE). Protocols for 3' RACE require the careful design and selection of nested primers within the 3' untranslated region (3' UTR) of the target gene of interest. However, with a few modifications the protocol can be used to include the entire 3' UTR and sequences within the open reading frame (ORF), providing a more comprehensive picture of the relationship between the ORF and the 3' UTR. This is in addition to identification of the polyadenylation signal (PAS), as well as the cleavage and polyadenylation site provided by conventional 3' RACE. Expanded 3' RACE can detect unusual 3' UTRs, including gene fusions within the 3' UTR, and the sequence information can be used to predict potential miRNA binding sites as well as AU rich destabilizing elements that may affect the stability of the transcript.

| S-EPMC5933262 | biostudies-literature

Simple and inexpensive three-step rapid amplification of cDNA 5' ends using 5' phosphorylated primers.

Project description:Rapid amplification of cDNA 5' ends (5'-RACE) is routinely used for the sequence analysis of the upstream noncoding regions of cellular mRNAs; however, it represents a tedious and cost-intensive procedure. By employing 5' phosphorylated gene-specific primers for first-strand cDNA synthesis, we cut short the previously established reverse ligation and amplification protocol of Mandl and coworkers (BioTechniques, 1991, vol. 10, pp. 484-486) to a streamlined three-step procedure that no longer depends on enzymatic mRNA decapping or linker ligation. The novel three-step protocol has been validated by mapping the transcriptional start sites of heterologously expressed yellow fever virus genomic RNAs from cultured mammalian cells.

| S-EPMC3562438 | biostudies-literature

Transcriptome profiling of Mesoplasma florum strain L1 using RNA sequencing (RNA-Seq) and genome-wide 5'-rapid amplification of cDNA ends (5'-RACE)

Project description:Mesoplasma florum is a near-minimal bacterium belonging to the Mollicutes class particularly characterized by its small genome, fast growth rate, and lack of pathogenic potential. Here, we report the first genome-wide transcriptomic analysis of this microorganism using directional RNA sequencing (RNA-Seq) and 5'-rapid amplification of cDNA ends (5'-RACE). 5'-RACE sequencing reads were used to identify 605 putative transcription start sites (TSSs), of which more than 400 could be associated to a conserved promoter sequence. Along with terminator predictions, these TSSs allowed the reconstruction of M. florum transcription units, covering more than 90% of annotated genes. We evaluated the expression level of all M. florum protein coding genes using paired-end RNA-Seq reads according to the number of fragments per kilobase per million of mapped reads (FPKM). FPKM values were converted to absolute transcript abundances using biomass quantification data. These efforts aim at reaching a deep understanding of global cellular mechanisms in M. florum and will guide future genome engineering projects in this simple organism.

2020-11-02 | GSE152985 | GEO

Mapping the 5' and 3' ends of Tetrahymena thermophila mRNAs using RNA ligase mediated amplification of cDNA ends (RLM-RACE).

Project description:A procedure is described for mapping the ends of RNAs. Using T4 RNA ligase, a DNA (3' end) or RNA (5' end) oligonucleotide is ligated to RNA ends followed by cDNA synthesis, PCR amplification, cloning and sequencing. This method determines 5' ends, 3' polyadenylation sites and the size of poly(A) tails, and should be applicable to non-polyadenylated mRNAs and to non-message RNAs. Analysis of four Tetrahymena thermophila histone mRNAs revealed multiple, closely spaced 5' ends consistent with those determined by other methods. Except for a 'CCAAT' box in either orientation 100-200 nucleotides upstream of the transcription start site, no conserved sequence elements were observed in the untranslated 5' region or in sequences immediately flanking the transcription start site. Analysis of the 3' ends of mRNAs encoding four histones, two tubulins and the Tetrahymena TATA binding protein confirmed the observations that Tetrahymena histone messages are polyadenylated and that poly(A) tails in this organism are short (approximately 50 nt). No canonical poly(A) addition signal was identified. The four histone messages analyzed have contained three sequence elements, TGTGT-TAA-AAGTATT, not found in non-histone messages. Two non-histone messages contained GCATT(N)15ATACC near the poly(A) addition site.

| S-EPMC311412 | biostudies-other

Identification and Sequencing of Candida krusei Aconitate Hydratase Gene Using Rapid Amplification of cDNA Ends Method and Phylogenetic Analysis.

Project description:BACKGROUND:The production and development of an effective fungicidal drug requires the identification of an essential fungal protein as a drug target. Aconitase (ACO) is a mitochondrial protein that plays a vital role in tricarboxylic acid (TCA) cycle and thus production of energy within the cell. OBJECTIVES:The current study aimed to sequence Candida krusei ACO gene and determine any amino acid residue differences between human and fungal aconitases to obtain selective inhibition. MATERIALS AND METHODS:Candida krusei (ATCC: 6258) aconitase gene was determined by 5'Rapid Amplification of cDNA Ends (RACE) method and degenerate Polymerase Chain Reaction (PCR) and analyzed using bioinformatics softwares. RESULTS:One thousand-four hundred-nineteen nucleotide of C. krusei aconitase gene were clarified and submitted in Genbank as a partial sequence and then taxonomic location of C. krusei was determined by nucleotide and amino acid sequences of this gene. The comparison of nucleotide and amino acid sequences of Candida species ACO genes showed that C. krusei possessed characteristic sequences. No significant differences were observed between C. krusei and human aconitases within the active site amino acid residues. CONCLUSIONS:Results of the current study indicated that aconitase was not a suitable target to design new anti-fungal drugs that selectively block this enzyme.

| S-EPMC4736022 | biostudies-literature

5' RACE (Rapid amplification of cDNA ends) and 3' RACE of U6 or U1-driven Cas13 CRISPR RNAs (crRNAs)

Project description:5' RACE (Rapid amplification of cDNA ends) and 3' RACE of U6 or U1-driven Cas13 CRISPR RNAs (crRNAs)

| PRJNA1150101 | ENA

5' Rapid Amplification of cDNA Ends and Illumina MiSeq Reveals B Cell Receptor Features in Healthy Adults, Adults With Chronic HIV-1 Infection, Cord Blood, and Humanized Mice.

Project description:Using 5' rapid amplification of cDNA ends, Illumina MiSeq, and basic flow cytometry, we systematically analyzed the expressed B cell receptor (BCR) repertoire in 14 healthy adult PBMCs, 5 HIV-1+ adult PBMCs, 5 cord blood samples, and 3 HIS-CD4/B mice, examining the full-length variable region of μ, γ, α, κ, and λ chains for V-gene usage, somatic hypermutation (SHM), and CDR3 length. Adding to the known repertoire of healthy adults, Illumina MiSeq consistently detected small fractions of reads with high mutation frequencies including hypermutated μ reads, and reads with long CDR3s. Additionally, the less studied IgA repertoire displayed similar characteristics to that of IgG. Compared to healthy adults, the five HIV-1 chronically infected adults displayed elevated mutation frequencies for all μ, γ, α, κ, and λ chains examined and slightly longer CDR3 lengths for γ, α, and λ. To evaluate the reconstituted human BCR sequences in a humanized mouse model, we analyzed cord blood and HIS-CD4/B mice, which all lacked the typical SHM seen in the adult reference. Furthermore, MiSeq revealed identical unmutated IgM sequences derived from separate cell aliquots, thus for the first time demonstrating rare clonal members of unmutated IgM B cells by sequencing.

| S-EPMC5879793 | biostudies-literature

Transcriptome profiling of Mesoplasma florum strain L1 using RNA sequencing (RNA-Seq) and genome-wide 5'-rapid amplification of cDNA ends (5'-RACE)

Project description:Transcriptome profiling of Mesoplasma florum strain L1 using RNA sequencing (RNA-Seq) and genome-wide 5'-rapid amplification of cDNA ends (5'-RACE)

| PRJNA641121 | ENA

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

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