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Global analysis of off-target genes by GAPDH siRNA

ABSTRACT: A GAPDH siRNA was overexpressed in HeLa cells and microarray expression profiling was performed to experimentally identify the off-targets of this siRNA at the transcriptome level. Among all predicted siRNA off-targets by MirTarget2, the majority were downregulated by at least 10% (i.e. less than 90% remaining gene expression). Moreover, more than one fifth were downregulated by at least 30%. The gene knockdown data is consistent with previous observations that siRNA off-targets are generally silenced at a more moderate level as compared to silencing of the single intended target GAPDH siRNA was transfected into HeLa cells, and the unintended off-targets was identified by microarrays 24 hours post transfection.

ORGANISM(S): Homo sapiens

SUBMITTER: Xiaowei Wang

PROVIDER: E-GEOD-41924 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Global analysis of off-target genes by GAPDH siRNA

Project description:A GAPDH siRNA was overexpressed in HeLa cells and microarray expression profiling was performed to experimentally identify the off-targets of this siRNA at the transcriptome level. Among all predicted siRNA off-targets by MirTarget2, the majority were downregulated by at least 10% (i.e. less than 90% remaining gene expression). Moreover, more than one fifth were downregulated by at least 30%. The gene knockdown data is consistent with previous observations that siRNA off-targets are generally silenced at a more moderate level as compared to silencing of the single intended target

2013-10-03 | GSE41924 | GEO

An uncharacterized gene, C7orf26, identified from an autosomal dominant ocular disease functions in mRNA splicing

Project description:An uncharacterized gene, C7orf26 was expected to function with the integrator complex in mRNA splicing. To address the involvement of mRNA splicing, we performed microarray analysis of HeLa/C7orf26 siRNA cells and HeLa/non-specific (NS) and identified 700 transcripts that were downregulated by 50% or more in C7orf26 siRNA-treated HeLa cells. Gene set enrichment analysis showed that genes tagged with ‘alternative splicing’ or ‘splice variant’ occupy a large fraction of the downregulated genes.

2023-03-08 | GSE186323 | GEO

siRNA off-target effects can be reduced at concentrations that match their individual potency

Project description:This study demonstrates that siRNA off-targets (e.g. 3'UTR off-targets), can be significantly reduced when cells are treated with a relatively low dose of siRNA (e.g. 1nM) that is sufficient to effectively silence the intended target.

2011-08-01 | GSE28786 | GEO

Homo sapiens

Project description:Gene profile in H1299 cells treated with PTD-DRBD GAPDH siRNA or treated with Lipofection GAPDH siRNA

| PRJNA116613 | ENA

siRNA of human HeLa cells reveals Staufen1 regulates a variety of mammalian transcripts

Project description:It is currently unknown how extensively the double-stranded RNA binding protein Staufen (Stau)1 is utilized by mammalian cells to regulate gene expression. To date, Stau1 binding to the 3â untranslated region (3âUTR) of ARF1 mRNA has been shown to target ARF1 mRNA for Stau1-mediated mRNA decay (SMD). ARF1 SMD depends on translation and recruitment of the nonsense-mediated mRNA decay factor Upf1 to the ARF1 3âUTR by Stau1. Here, we use microarray analyses to examine changes in the abundance of cellular mRNAs that occur when Stau1 is depleted. Results indicate that 1.1% and 1.0% of the 11,569 HeLa-cell transcripts that were analyzed are, respectively, upregulated and downregulated at least two-fold in three independently performed experiments. Additionally, we localize the Stau1 binding site to the 3âUTR of four mRNAs that we define as natural SMD targets. Together, these and substantiating results suggest that Stau1 influences the expression of a wide variety of physiologic transcripts and metabolic pathways. Experiment Overall Design: We report the results of three independently performed microarray analyses that examined changes in the abundance of transcripts from HeLa-cell genes upon Stau1 depletion. HeLa cells were transiently transfected with either a nonspecific Control small interfering (si)RNA or Stau1 siRNA. Stau1 siRNA reduced the level of cellular Stau1 to as little as 4% of normal, where normal is defined as the level in the presence of Control siRNA (data not shown). RNA from three independently performed transfections was separately hybridized to microarrays.

2008-06-15 | E-GEOD-6679 | biostudies-arrayexpress

Widespread siRNA "off-target" transcript silencing mediated by seed region sequence complementarity

Project description:Transfected siRNAs and miRNAs regulate numerous transcripts that have only limited complementarity to the active strand of the RNA duplex. This process reflects natural target regulation by miRNAs, but is an unintended (“off-target”) consequence of siRNA-mediated silencing. Here we demonstrate that this unintended off-target silencing is widespread, and occurs in a manner reminiscent of target silencing by miRNAs. A high proportion of unintended transcripts silenced by siRNAs showed 3’ UTR sequence complementarity to the seed region of the siRNA. Base mismatches within the siRNA seed region reduced the set of original off-target transcripts but generated new sets of silenced transcripts with sequence complementarity to the mismatched seed sequence. An inducible shRNA silenced a subset of transcripts that were silenced by an siRNA of the same sequence, demonstrating that unintended silencing is sequence-mediated and is independent of delivery method. In all cases, off-target transcript silencing was accompanied by loss of the corresponding protein and occurred with similar dependence on siRNA concentration as silencing of the target transcript. These results demonstrate that short stretches of sequence complementarity to the seed region of the siRNA are key to the silencing of unintended transcripts, and that this limits the specificity of siRNA-mediated transcript silencing. Because these off-target events are sequence-dependent, inclusion of multiple independent siRNAs to the target of interest can help to distinguish true positives from false positives in functional genetic analyses. Keywords: siRNA, RNAi, sequence alignment, off-target, seed region

2006-09-12 | GSE5814 | GEO

Reduced seed region-based off-target activity with lentivirus-mediated RNAi

Project description:In addition to silencing intended target genes, transfected siRNAs regulate numerous unintended transcripts through a mechanism in which the equivalent of a microRNA-like seed region in the siRNA recognizes complementary sequences in transcript 3' UTRs. The ability to limit such off-target effects would substantially facilitate accurate interpretation of RNA interference (RNAi) experiments and thus greatly enhance their value. We tested whether lentivirus-mediated delivery of shRNA is prone to the seed region-based off-target activity prevalent in siRNA experiments. We compared target gene silencing and overall impact on global gene expression caused by multiple 21-mer duplex sequences delivered as both transfected siRNA and lentivirus vector-expressed shRNA. At equivalent levels of target gene silencing, signatures induced by shRNAs were significantly smaller than those induced by cognate siRNAs and arose less frequently from seed region activity. Interestingly, the low level of seed-region based off-target activity exhibited by shRNAs resulted in down-regulation of transcripts that were largely distinct from those regulated by siRNAs. On the basis of these observations, we recommend lentivirus-mediated RNA interference for pathway profiling experiments that measure whole genome transcriptional readouts as well as for large-scale screens when resources for extensive follow up are limited.

2010-06-09 | GSE22209 | GEO

Widespread siRNA "off-target" transcript silencing mediated by seed region sequence complementarity

Project description:Transfected siRNAs and miRNAs regulate numerous transcripts that have only limited complementarity to the active strand of the RNA duplex. This process reflects natural target regulation by miRNAs, but is an unintended (â??off-targetâ??) consequence of siRNA-mediated silencing. Here we demonstrate that this unintended off-target silencing is widespread, and occurs in a manner reminiscent of target silencing by miRNAs. A high proportion of unintended transcripts silenced by siRNAs showed 3â?? UTR sequence complementarity to the seed region of the siRNA. Base mismatches within the siRNA seed region reduced the set of original off-target transcripts but generated new sets of silenced transcripts with sequence complementarity to the mismatched seed sequence. An inducible shRNA silenced a subset of transcripts that were silenced by an siRNA of the same sequence, demonstrating that unintended silencing is sequence-mediated and is independent of delivery method. In all cases, off-target transcript silencing was accompanied by loss of the corresponding protein and occurred with similar dependence on siRNA concentration as silencing of the target transcript. These results demonstrate that short stretches of sequence complementarity to the seed region of the siRNA are key to the silencing of unintended transcripts, and that this limits the specificity of siRNA-mediated transcript silencing. Because these off-target events are sequence-dependent, inclusion of multiple independent siRNAs to the target of interest can help to distinguish true positives from false positives in functional genetic analyses. For details, please see A.L. Jackson, et al. 2006. RNA 12(7): 1179-1187. We used consensus genelists for clustering. Please see attached tables for genelists.

2006-09-12 | E-GEOD-5814 | biostudies-arrayexpress

Homo sapiens

Project description:Hela SPAG4 siRNA

| PRJNA197141 | ENA

microRNA-like off-target transcript regulation by siRNAs

Project description:siRNAs mediate sequence-specific gene silencing in cultured mammalian cells but also silence unintended transcripts. Many siRNA off-target transcripts match the guide-strand ‘‘seed region,’’ similar to the way microRNAs match their target sites. The extent to which this seed-matched, microRNA-like, off-target silencing affects the specificity of therapeutic siRNAs in vivo is currently unknown. Here, we compare microRNA-like off-target regulations in mouse liver in vivo with those seen in cell culture for a series of therapeutic candidate siRNAs targeting Apolipoprotein B (APOB). Each siRNA triggered regulation of consistent microRNA-like off-target transcripts in mouse livers and in cultured mouse liver tumor cells. In contrast, there was only random overlap between microRNA-like off-target transcripts from cultured human and mouse liver tumor cells. Therefore, siRNA therapeutics may trigger microRNA-like silencing of many unintended targets in vivo, and the potential toxicities caused by these off-target gene regulations cannot be accurately assessed in rodent models.

2009-02-01 | GSE14073 | GEO

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