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Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Global gene expression profiling of mouse liver tissue samples after treated with various anti-HCV shRNAs

ABSTRACT: RNA interference (RNAi) is becoming routine in scientific discovery and treating human disease. However, its applications are hampered by unwanted effects. The RNA-Seq analyses from mouse liver samples expressing various anti-HCV shRNAs show that weak base-pairing in both seed and 3’ regions is required to achieve minimal off-targeting effect. We created nine shRNAs against the HCV genome, and transfected shRNA-expressing plasmids into mouse liver via a hydrodynamic tail vein infusion. A plasmid backbone lacking the shRNA sequence was used as a negative control. After 7 days, mouse liver RNAs were harvested and subject to RNA-Seq analysis (two biological repeats).

ORGANISM(S): Mus musculus

SUBMITTER: Yue Zhang

PROVIDER: E-GEOD-55131 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Weak base pairing in both seed and 3' regions reduces RNAi off-targets and enhances si/shRNA designs.

Gu Shuo S Zhang Yue Y Jin Lan L Huang Yong Y Zhang Feijie F Bassik Michael C MC Kampmann Martin M Kay Mark A MA

Nucleic acids research 20140930 19

The use of RNA interference is becoming routine in scientific discovery and treatment of human disease. However, its applications are hampered by unwanted effects, particularly off-targeting through miRNA-like pathways. Recent studies suggest that the efficacy of such off-targeting might be dependent on binding stability. Here, by testing shRNAs and siRNAs of various GC content in different guide strand segments with reporter assays, we establish that weak base pairing in both seed and 3' region ...[more]

PMID: 25270879

Similar Datasets

Global gene expression profiling of mouse liver tissue samples after treated with various anti-HCV shRNAs

Project description:RNA interference (RNAi) is becoming routine in scientific discovery and treating human disease. However, its applications are hampered by unwanted effects. The RNA-Seq analyses from mouse liver samples expressing various anti-HCV shRNAs show that weak base-pairing in both seed and 3’ regions is required to achieve minimal off-targeting effect.

2014-10-03 | GSE55131 | GEO

The loop position of shRNAs and pre-miRNAs is critical for the accuracy of Dicer processing in vivo

Project description:Short-hairpin RNA (shRNA)-induced RNAi is used for biological discovery and therapeutics. Dicer, whose normal role is to liberate endogenous miRNAs from their precursors, processes shRNAs into different biologically active siRNAs, affecting their efficacy and potential for off-targeting. We found that in cells, Dicer induced imprecise cleavage events around the expected sites based on the previously described 5'/3'-counting rules. These promiscuous non-canonical cleavages were abrogated when the cleavage site was positioned 2 nt from a bulge or loop. Interestingly, we observed that the ~1/3 of mammalian endogenous pre-miRNAs that contained such structures were more precisely processed by Dicer. Implementing a new "loop-counting rule", we designed potent anti-HCV shRNAs with substantially reduced off-target effects. Our results suggest that Dicer recognizes the loop/bulge structure in addition to the ends of shRNAs/pre-miRNAs for accurate processing. This has important implications for both miRNA processing and future design of shRNAs for RNAi-based genetic screens and therapies.

2012-12-25 | GSE41292 | GEO

shRNA Sensor Analysis of DSIR Selected shRNAs

Project description:Purpose: Generate a large, high quality database of paired shRNA efficacy/sequence datapoints. Methods:Twelve shRNAs for each Refseq annotated human gene were selected based on the DSIR algorithm. Twelve batches of ~22K shRNAs (corresponding to 12 agilent chips) were then assessed for efficacy via the sensor method outlined in Fellmann et al, Mol Cell, 2011. Conclusions: Neighboring nucleotide combinations are best at predicting shRNA efficacy.

2014-11-24 | E-GEOD-62183 | biostudies-arrayexpress

The loop position of shRNAs and pre-miRNAs is critical for the accuracy of Dicer processing in vivo

2012-12-25 | E-GEOD-41292 | biostudies-arrayexpress

Genome-wide expression profiling of cells infected with control or RPS14 shRNAs

Project description:We performed genome-wide expression profiling of cells infected with control or RPS14 shRNAs. Keywords: controls vs. shRNA treated

2008-01-17 | GSE9487 | GEO

Sub-genomewide shRNAs constructed using an optimized selection algorithm and microRNA backbone provide stronger evidence for follow-up studies

Project description:Sub-genomewide shRNA libraries were constructed using the current RNAi consortium constructs as well as using the DSIR (siRNA algoirthm) and a novel shRNA specific algorithm (shERWOOD). All libraries were placed into mir30 expression vectors. The shERWOOD libraries were also placed in a vector harboring an optimized mir cassette (ultramir). Each library was screened using the pancreatic cell line A385. A concensus set of essential genes identified as the set for which two shRNAs depleted in each of the libries. For these genes, a great percentage of shERWOOD seletected shRNA depleted. In addition the placement of shERWOOD selected constructs into ultramir scaffoled increased the rate of shRNA depletion for essential genes further. Purpose: shRNA screens were carried out using various library construction strategies to identify the strategy that provides the best shRNA screening results. Method: Libraries were constructed using the TRC shRNA set as well as shRNAs identified using the DSIR and shERWOOD algorithms. shRNA libraries were cloned into mir30 expression vectors. shERWOOD shRNAs were also cloned into an expression vector harboring an optimized microRNA scaffold termed ultramir. Each resultant library was screened using the pancreatic cell line A385. Each library was analyzed separately to identify a set of genes where at least two shRNAs depleted. These gene sets were intersected to develop a set of essential genes. Results: The shERWOOD shRNA libraries provided the highest number depleting shRNAs for each essential gene. Further these shRNAs depleted to a greater extent than did the shRNAs from the other libraries. When shERWOOD libraries were placed into the ultramir cassette a greater number of shRNAs per essential gene depleted.

2014-11-24 | E-GEOD-62185 | biostudies-arrayexpress

LNCaP prostate cancer cells stably transfected with shRNAs targeting ADRB2

Project description:Gene expression profiling of prostate adenocarcinoma LNCaP cells stably transfected with shRNAs targeting ADRB2 (shADRB2-2 and shADRB2-3) or control shRNA (shCtrl) incubated in 10% FCS.

2019-04-25 | GSE130204 | GEO

shRNA Sensor Analysis of 5' U converted shRNAs reveals 3' target bias exists in addition to 5' guide bias

Project description:shRNAs selected with the shERWOOD algorithm were converted to have a U at the 5' end of their guide. When endogenous 1U shRNAs were compared to artificial shRNA via the sensor algorithm, the endogenous shRNAs were found to be more efficacious. Purpose: Structural studies have hinted that the 5' end of shRNA guides is engulfed in the RISC complex. It has also been reported that shRNAs with a 5' U are more efficacious than those with other 5' caps. We wished to determine whether replacement of shRNA guide 5' nucleotides with a U, regardless of the corresponding target base, would increase their efficacy. Method: For each gene in the "druggable genome" 10 shRNAs were selected with the shERWOOD algorithm. In each case the score was assessed as if the guide had a 5' U. Sensor constructs were designed pairing 1U-guide shRNAs with their endogenous target. shRNAs were assessed for efficacy via the shRNA sensor assay (Fellmann et al. Mol Cell 2011). Results: shRNAs with artificial 5' Us were found to be less efficacious than those with an endogenous 5' U,

2014-11-24 | E-GEOD-62186 | biostudies-arrayexpress

shRNA Sensor Analysis of 5' U converted shRNAs reveals 3' target bias exists in addition to 5' guide bias

2014-11-24 | GSE62186 | GEO

Expression data from Hodgkin lymphoma cell lines UHO-1 and L-1236 transduced with shRNAs against GATA-3 or non-functional control shRNAs

Project description:The transcription factor network in Hodgkin lymphoma (HL) represents a unique composition of proteins found in no other hematopoietic cell. Among these factors, an aberrant expression of the T cell transcription factor GATA-3 is observed in the B cell-derived Hodgkin and Reed/Sternberg (HRS) tumor cells. Herein, we elucidated the regulation and function of this factor in HL To obtain a more comprehensive overview of GATA-3 regulated genes in cHL cell lines, we performed a gene chip analysis comparing gene expression in GATA-3-specific shRNA-transduced L-1236 and U-HO1 cells compared to samples transduced with vectors encoding scrambled shRNAs. 12 Total samples were analyzed. Three independent samples were analyzed per condition and cell line.We generated the following pairwise comparisons using Bioconductor: shRNA Gata-3 UHO-1 < control shRNA UHO-1; shRNA Gata-3 L1236 < control shRNA L-1236. Genes with an FDR≤ 25% and a fold-change ≥ 1.5 and ≤ -1.5 were selected.

2010-07-24 | E-GEOD-22550 | biostudies-arrayexpress

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