scholarly journals Large-Scale, High-Throughput Validation of Short Hairpin RNA Sequences for RNA Interference

2006 ◽  
Vol 11 (3) ◽  
pp. 236-246 ◽  
Author(s):  
Laurence H. Lamarcq ◽  
Bradley J. Scherer ◽  
Michael L. Phelan ◽  
Nikolai N. Kalnine ◽  
Yen H. Nguyen ◽  
...  
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
Strong Support ◽  
Gc Content ◽  
Rapid Identification ◽  
Hairpin Rna ◽  
Rna Sequences ◽  
Short Hairpin ◽  
Short Hairpin Rnas ◽  
Interfering Rna

A method for high-throughput cloning and analysis of short hairpin RNAs (shRNAs) is described. Using this approach, 464 shRNAs against 116 different genes were screened for knockdown efficacy, enabling rapid identification of effective shRNAs against 74 genes. Statistical analysis of the effects of various criteria on the activity of the shRNAs confirmed that some of the rules thought to govern small interfering RNA (siRNA) activity also apply to shRNAs. These include moderate GC content, absence of internal hairpins, and asymmetric thermal stability. However, the authors did not find strong support for positionspecific rules. In addition, analysis of the data suggests that not all genes are equally susceptible to RNAinterference (RNAi).

scholarly journals Optimized RNA-targeting CRISPR/Cas13d technology outperforms shRNA in identifying functional circRNAs

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Yang Zhang ◽  
Tuan M. Nguyen ◽  
Xiao-Ou Zhang ◽  
Limei Wang ◽  
Tin Phan ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
High Throughput ◽  
Biological Effect ◽  
Human Hepatocellular Carcinoma ◽  
Guide Rnas ◽  
Rna Targeting ◽  
Short Hairpin ◽  
Short Hairpin Rnas ◽  
Screening Library ◽  
High Throughput Study

AbstractShort hairpin RNAs (shRNAs) are used to deplete circRNAs by targeting back-splicing junction (BSJ) sites. However, frequent discrepancies exist between shRNA-mediated circRNA knockdown and the corresponding biological effect, querying their robustness. By leveraging CRISPR/Cas13d tool and optimizing the strategy for designing single-guide RNAs against circRNA BSJ sites, we markedly enhance specificity of circRNA silencing. This specificity is validated in parallel screenings by shRNA and CRISPR/Cas13d libraries. Using a CRISPR/Cas13d screening library targeting > 2500 human hepatocellular carcinoma-related circRNAs, we subsequently identify a subset of sorafenib-resistant circRNAs. Thus, CRISPR/Cas13d represents an effective approach for high-throughput study of functional circRNAs.

scholarly journals Technological advancements and their importance for nematode identification

SOIL ◽  
2016 ◽  
Vol 2 (2) ◽  
pp. 257-270 ◽  
Author(s):  
Mohammed Ahmed ◽  
Melanie Sapp ◽  
Thomas Prior ◽  
Gerrit Karssen ◽  
Matthew Alan Back
Keyword(s):  
High Throughput ◽  
Crop Production ◽  
Large Scale ◽  
High Throughput Sequencing ◽  
Biological Indicators ◽  
Rapid Identification ◽  
Community Studies ◽  
Terrestrial Environments ◽  
Traditional Taxonomy ◽  
Sequencing Platforms

Abstract. Nematodes represent a species-rich and morphologically diverse group of metazoans known to inhabit both aquatic and terrestrial environments. Their role as biological indicators and as key players in nutrient cycling has been well documented. Some plant-parasitic species are also known to cause significant losses to crop production. In spite of this, there still exists a huge gap in our knowledge of their diversity due to the enormity of time and expertise often involved in characterising species using phenotypic features. Molecular methodology provides useful means of complementing the limited number of reliable diagnostic characters available for morphology-based identification. We discuss herein some of the limitations of traditional taxonomy and how molecular methodologies, especially the use of high-throughput sequencing, have assisted in carrying out large-scale nematode community studies and characterisation of phytonematodes through rapid identification of multiple taxa. We also provide brief descriptions of some the current and almost-outdated high-throughput sequencing platforms and their applications in both plant nematology and soil ecology.

scholarly journals Viral Vectors Applied for RNAi-Based Antiviral Therapy

Viruses ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 924 ◽  
Author(s):  
Kenneth Lundstrom
Keyword(s):  
Antiviral Therapy ◽  
Viral Vectors ◽  
Rna Virus ◽  
Micro Rna ◽  
Attractive Alternative ◽  
Rna Modifications ◽  
Hairpin Rna ◽  
Pathogenic Viruses ◽  
Short Hairpin ◽  
Interfering Rna

RNA interference (RNAi) provides the means for alternative antiviral therapy. Delivery of RNAi in the form of short interfering RNA (siRNA), short hairpin RNA (shRNA) and micro-RNA (miRNA) have demonstrated efficacy in gene silencing for therapeutic applications against viral diseases. Bioinformatics has played an important role in the design of efficient RNAi sequences targeting various pathogenic viruses. However, stability and delivery of RNAi molecules have presented serious obstacles for reaching therapeutic efficacy. For this reason, RNA modifications and formulation of nanoparticles have proven useful for non-viral delivery of RNAi molecules. On the other hand, utilization of viral vectors and particularly self-replicating RNA virus vectors can be considered as an attractive alternative. In this review, examples of antiviral therapy applying RNAi-based approaches in various animal models will be described. Due to the current coronavirus pandemic, a special emphasis will be dedicated to targeting Coronavirus Disease-19 (COVID-19).

scholarly journals Short Hairpin RNAs for Strand-Specific Small Interfering RNA Production

2020 ◽  
Vol 8 ◽  
Author(s):  
Peike Sheng ◽  
Krystal A. Flood ◽  
Mingyi Xie
Keyword(s):  
Small Interfering Rna ◽  
Short Hairpin ◽  
Short Hairpin Rnas ◽  
Interfering Rna

Rapid Construction of Small Interfering RNA-Expressing Adenoviral Vectors on the Basis of Direct Cloning of Short Hairpin RNA-Coding DNAs

2007 ◽  
Vol 18 (1) ◽  
pp. 74-80 ◽  
Author(s):  
Hiroyuki Mizuguchi ◽  
Naoko Funakoshi ◽  
Tetsuji Hosono ◽  
Fuminori Sakurai ◽  
Kenji Kawabata ◽  
...  
Keyword(s):  
Small Interfering Rna ◽  
Adenoviral Vectors ◽  
Short Hairpin Rna ◽  
Hairpin Rna ◽  
Direct Cloning ◽  
Rapid Construction ◽  
Short Hairpin ◽  
Interfering Rna

scholarly journals The Kinesin KIF1C and Microtubule Plus Ends Regulate Podosome Dynamics in Macrophages

2006 ◽  
Vol 17 (6) ◽  
pp. 2811-2823 ◽  
Author(s):  
Petra Kopp ◽  
Reiner Lammers ◽  
Martin Aepfelbacher ◽  
Günther Woehlke ◽  
Thomas Rudel ◽  
...  
Keyword(s):  
Molecular Basis ◽  
Small Interfering Rna ◽  
Migration And Invasion ◽  
Nonmuscle Myosin ◽  
Subcellular Targeting ◽  
Hairpin Rna ◽  
Monocytic Cells ◽  
Human Macrophages ◽  
Short Hairpin ◽  
Interfering Rna

Microtubules are important for the turnover of podosomes, dynamic, actin-rich adhesions implicated in migration and invasion of monocytic cells. The molecular basis for this functional dependency, however, remained unclear. Here, we show that contact by microtubule plus ends critically influences the cellular fate of podosomes in primary human macrophages. In particular, we identify the kinesin KIF1C, a member of the Kinesin-3 family, as a plus-end–enriched motor that targets regions of podosome turnover. Expression of mutation constructs or small interfering RNA-/short hairpin RNA-based depletion of KIF1C resulted in decreased podosome dynamics and ultimately in podosome deficiency. Importantly, protein interaction studies showed that KIF1C binds to nonmuscle myosin IIA via its PTPD-binding domain, thus providing an interface between the actin and tubulin cytoskeletons, which may facilitate the subcellular targeting of podosomes by microtubules. This is the first report to implicate a kinesin in podosome regulation and also the first to describe a function for KIF1C in human cells.

scholarly journals Structural Features of MicroRNA (miRNA) Precursors and Their Relevance to miRNA Biogenesis and Small Interfering RNA/Short Hairpin RNA Design

2004 ◽  
Vol 279 (40) ◽  
pp. 42230-42239 ◽  
Author(s):  
Jacek Krol ◽  
Krzysztof Sobczak ◽  
Urszula Wilczynska ◽  
Maria Drath ◽  
Anna Jasinska ◽  
...  
Keyword(s):  
Small Interfering Rna ◽  
Structural Features ◽  
Short Hairpin Rna ◽  
Mirna Biogenesis ◽  
Hairpin Rna ◽  
Short Hairpin ◽  
Rna Design ◽  
Interfering Rna

scholarly journals Optimized RNA-targeting CRISPR/Cas13d technology outperforms shRNA in identifying essential circRNAs

2020 ◽  
Author(s):  
Yang Zhang ◽  
Tuan M. Nguyen ◽  
Xiao-Ou Zhang ◽  
Tin Phan ◽  
John G. Clohessy ◽  
...  
Keyword(s):  
High Throughput ◽  
Biological Effect ◽  
High Rate ◽  
Circular Rnas ◽  
Hairpin Rna ◽  
Guide Rnas ◽  
Rna Targeting ◽  
Bona Fide ◽  
Short Hairpin ◽  
High Throughput Manner

AbstractCircular RNAs (circRNAs) are widely expressed, but their functions remain largely unknown. To study circRNAs in a high-throughput manner, short hairpin RNA (shRNA) screens1 have recently been used to deplete circRNAs by targeting their unique back-splicing junction (BSJ) sites. Here, we report frequent discrepancies between shRNA-mediated circRNA knockdown efficiency and the corresponding biological effect, raising pressing concerns about the robustness of shRNA screening for functional circRNAs. To address this issue, we leveraged the CRISPR/Cas13d system2 for circRNAs functional screenings. We optimized a strategy for designing single guide RNAs to deplete circRNAs. We then performed shRNA and CRISPR/Cas13d parallel screenings and demonstrated that shRNA-mediated circRNAs screening yielded a high rate of false positives phenotypes, while optimized CRISPR/Cas13d led to the identification of bona-fide functional circRNAs. Collectively, we developed a specific and reliable approach to functionalize circRNAs in a high-throughput manner.

Rapid Construction of Small Interfering RNA-Expressing Adenoviral Vectors on the Basis of Direct Cloning of Short Hairpin RNA-Coding DNAs

2006 ◽  
Vol 0 (0) ◽  
pp. 061222104941001
Author(s):  
Hiroyuki Mizuguchi ◽  
Naoko Funakoshi ◽  
Tetsuji Hosono ◽  
Fuminori Sakurai ◽  
Kenji Kawabata ◽  
...  
Keyword(s):  
Small Interfering Rna ◽  
Adenoviral Vectors ◽  
Short Hairpin Rna ◽  
Hairpin Rna ◽  
Direct Cloning ◽  
Rapid Construction ◽  
Short Hairpin ◽  
Interfering Rna

High-throughput function-based gene identification using enzymatically generated short hairpin RNA library and massive parallel sequencing

2010 ◽  
Vol 27 ◽  
pp. S37
Author(s):  
M. Shtutman ◽  
A. Maliyekkel ◽  
E. Levina ◽  
P. Ohouo ◽  
Y. Shao ◽  
...  
Keyword(s):  
High Throughput ◽  
Short Hairpin Rna ◽  
Massive Parallel Sequencing ◽  
Gene Identification ◽  
Hairpin Rna ◽  
Parallel Sequencing ◽  
Short Hairpin
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