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Real-time quantitative PCR analysis of miRNA expression in human melanoma MMRU cell line after Sox4 knockdown

ABSTRACT: We used Applied Biological Materials (ABM)’s miRNA profiling platform to quantitate expression of miRNAs in a human melanoma cell line (MMRU) in Sox4 siRNA treated cells (siSox4) compared with the control siRNA treated cells (siCTR) or after overexpression of Flag-Dicer in Sox4 knockdown cells (siSox4=F-Dicer) compared with control cells. 72 hours after transfection, Total RNA from siCTR, siCTR+empty Flag-vector (CTR), siSox4 or siSox4+-F-Dicer transfected MMRU cells was prepared with Qiazol extraction followed by Poly-A Tailing reactions and miRNA cDNA synthesis (ABM C204). Real-time qPCR reactions and instrumental analysis was performed using Roche LightCycler480.

ORGANISM(S): Homo sapiens

SUBMITTER: Seyed Mehdi Jafarnejad

PROVIDER: E-GEOD-36715 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Real-time quantitative PCR analysis of miRNA expression in human melanoma MMRU cell line after Sox4 knockdown

Project description:We used Applied Biological Materials (ABM)’s miRNA profiling platform to quantitate expression of miRNAs in a human melanoma cell line (MMRU) in Sox4 siRNA treated cells (siSox4) compared with the control siRNA treated cells (siCTR) or after overexpression of Flag-Dicer in Sox4 knockdown cells (siSox4=F-Dicer) compared with control cells.

2012-06-01 | GSE36715 | GEO

Transcriptome wide identification of Dicer binding in human and C. elegans reveals a variety of substrates (small RNA AGO-IP)

Project description:Dicer is a deeply conserved endoribonuclease with key functions in small RNA biogenesis. Here we employed PAR-CLIP/iPAR-CLIP to identify direct Dicer binding sites in the transcriptomes of human cells and human. We found hundreds of novel miRNAs and non-canonical Dicer substrates with high sensitivity. Small RNA production depended on structure of the binding site and is globally biased towards the 5' arm of hairpins. Unexpectedly, in both species Dicer bound numerous hairpins inside mRNAs without observable small RNA production. Our data revealed ~100 mRNAs of protein coding genes to be targeted in both human and worm. These mRNAs significantly overlapped with the RNAi pathway. We also, unexpectedly, found that mitochondrial transcripts are Dicer targets in both species. We demonstrate functional consequences of Dicer binding by perturbation analysis. Taken together,we provide the first genome-wide catalog of direct Dicer targets. Our results suggest widespread function outside of miRNA biogenesis. Argonaute loaded small RNAs were extracted from FLAG:AGO2 and FLAG:AGO3 expressing HEK293 cells. Small RNA was purified and length selected (see supplementary methods).

2014-11-20 | E-GEOD-55331 | biostudies-arrayexpress

SOX4 genome binding targets in glioblastoma cell line

Project description:Overexpression of SOX4 in LN229 glioblastoma cells prevents their cell cycle Examination of SOX4 binding profile in prostate cell LN229-SOX4 with LN229-GFP as negative control.

2014-10-20 | E-GEOD-51663 | biostudies-arrayexpress

A variety of Dicer substrates in human and C. elegans (HEK small RNA)

Project description:The endoribonuclease Dicer is known for its central role in the biogenesis of eukaryotic small RNAs/microRNAs. Despite its importance, Dicer target transcripts have not been directly mapped. Here, we apply biochemical methods to human cells and C. elegans and identify thousands of Dicer binding sites. We find known and hundreds of novel miRNAs with high sensitivity and specificity. We also report structural RNAs, promoter RNAs, and mitochondrial transcripts as Dicer targets. Interestingly, most Dicer binding sites reside on mRNAs/lncRNAs and are not significantly processed into small RNAs. These passive sites typically harbor small, Dicer-bound hairpins within intact transcripts and generally stabilize target expression. We show that passive sites can sequester Dicer and reduce microRNA expression. mRNAs with passive sites were in human and worm significantly associated with processing-body/granule function. Together, we provide the first transcriptome-wide map of Dicer targets and suggest conserved binding modes and functions outside the miRNA pathway. small RNAs were extracted from total RNA. Standard small RNA sequencing was performed to sequence 5' monophosphate bearing small RNAs. This was performed for siRNA knockdowns of DICER1 or DROSHA, with two independent siRNAs each, and for control cells. (5 samples in total)

2014-11-20 | E-GEOD-63458 | biostudies-arrayexpress

Genes regulated by SOX4 in glioblastoma cell line

Project description:Overexpression of SOX4 in LN229 glioblastoma cells prevents their cell cycle We used microarrays to detail the global programme of gene expression in SOX4 overexpression LN229 cells compared with mock control LN229 cells SOX4 overexpression LN229 cells and and control LN229 cells were cultured in DMEM cell culture media for RNA extraction and hybridization on Affymetrix microarrays. We sought to obtain the genes regulated by SOX4 in glioblastoma cell lines.

2014-09-30 | E-GEOD-51301 | biostudies-arrayexpress

DICER-interacting partners in juvenile mouse testes

Project description:DICER has a well-characterized role in the processing of microRNAs (miRNAs) and small interfering RNAs (siRNA) that are important for post-transcriptional gene regulation. Emerging evidence suggests that DICER also has several non-canonical functions beyond miRNA/siRNA biogenesis, for example in transcriptional gene silencing at the chromatin level, as well as in RNA degradation and maintenance of genomic integrity. We have shown that the function of DICER in germ cells is essential for normal spermatogenesis; male mice lacking DICER in postnatal male germ cells are infertile due to severe defects in haploid differentiation. To better understand the function of DICER in male germ cells, we immunoprecipitated DICER from juvenile mouse testes and performed mass spectrometric analysis to identify DICER-interacting proteins.

2020-06-03 | PXD017772 | Pride

Control of agricultural traits by hc-siRNA associated MITEs in rice

Project description:Transposable elements (TEs) and repetitive sequences comprise over 40% of rice genome. Different TEs are tightly regulated by distinct epigenetic mechanisms. For example, the activities of LTR retrotransposon Tos17 and non-LTR retrotransposon LINE element Karma are uniquely regulated by histone H3K9 methylation and histone H3K4 demethylation, respectively. Miniature inverted repeat transposable elements (MITEs) are one of the most high-copy-number DNA transposons, which are interspersed around rice genome and might influence nearby gene expression. In plants, 24-nucleotide (24-nt) heterochromatic small interfering RNAs (hc-siRNAs) derived from repeats and TEs. To what extent hc-siRNA associated TEs affect gene expression and therefore contribute to agricultural traits in rice remains elusive. Here, we show that OsDCL3a, one of Dicer-Like 3 (DCL3) homolog, is primarily responsible for 24-nt hc-siRNA processing in rice. Impaired OsDCL3a displayed altered important agricultural traits in rice. We found that genome-wide (281,563) 24-nt hc-siRNA clusters were OsDCL3a-dependent, among which MITEs were significantly enriched. Impaired OsDCL3a caused significant overlapping between reduced hc-siRNAs from MITEs and elevated nearby gene expression. Intriguingly, genes involved in Gibberellin and Brassinosteroid homeostasis were identified as direct targets of OsDCL3a, which may attribute to dwarfism and enlarged flag leaf angle upon OsDCL3a deficiency. Our work uncovers OsDCL3a-dependent hc-siRNAs derived from MITEs as broad spectrum of regulators for fine-tuning gene expression, and this observation may reflect a conserved mechanism in other higher plants with dispersed repeat- or TE-rich genomes. Examination of OsDCL3a-dependent hc-siRNAs derived from MITEs.

2014-02-04 | E-GEOD-50778 | biostudies-arrayexpress

Integrative AUF1 PAR-CLIP analysis uncovers AUF1 roles in translation and genome integrity (PAR-CLIP)

Project description:We report that AUF1 modulates global mRNA stability and translation, in turn promoting the maintenance of DNA integrity. Please see individual series. In short, for AUF1 PAR-CLIP, the four isoforms of AUF1 (p37, p40, p42, and p45) tagged with a Flag epitope were expressed in HEK293 cells. For total RNA-Seq HEK293 cells were transfected with Control siRNA, AUF1 siRNA, Empty Vector, Flag-AUF1 p37, p40, p42, or p45 as well as WI-38 cells were collected at PDL 15 and 55 and also transfected with Control siRNA, AUF1 siRNA, HuR siRNA. For Ribo-Seq HeLa cells were transfected with Control siRNA, AUF1 siRNA, or HuR siRNA.

2014-11-05 | E-GEOD-52971 | biostudies-arrayexpress

Expression profiling by array in ATLL

Project description:Previously, we have shown that an AP-1 family member Fra-2, which is hardly expressed in normal mature T cells, is consistently over-expressed in adult T-cell leukemia/lymphoma (ATLL), and together with JunD, upregulates CCR4 and many other genes including proto-oncogenes c-Myb, MDM2, Bcl-6, and SOX4. SOX4 is frequently overexpressed in many solid tumors and considered to be a potential oncogene. To identify downstream target genes of SOX4 and analyze SOX4 oncogenic pathway in ATLL , we compared gene expression profiles of ST1 cells transfected with SOX4 siRNA or control siRNA using the Affymetrix high density oligonucleotide microarray. The ATL-derived cell line ST1 was transfected with control siRNA or SOX4 siRNA using NucleofectorM-BM-. (Lonza, Basel, Switzerland). To determine the high-viability of the cells transfected with siRNAs, the cells were counted after 6 h on FACSCalibur (Becton Dickinson, Mountain View, CA) by gating out cells stained with propidium iodide. The transfection efficiency of RNAs was close to 95%, as determined by using fluorescent siRNA (Qiagen). After 48 h, total RNA samples were prepared and confirmed to be of good quality with the Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany). In addition, quantitative PCR confirmed that SOX4 siRNA effectively reduced SOX4 mRNA in ST1 cells.

2013-05-16 | E-GEOD-38021 | biostudies-arrayexpress

Integrative AUF1 PAR-CLIP analysis uncovers AUF1 roles in translation and genome integrity (RNA-Seq 1)

Project description:We report that AUF1 modulates global mRNA stability and translation, in turn promoting the maintenance of DNA integrity. Please see individual series. For AUF1 PAR-CLIP, the four isoforms of AUF1 (p37, p40, p42, and p45) tagged with a Flag epitope were expressed in HEK293 cells. For total RNA-Seq HEK293 cells were transfected with Control siRNA, AUF1 siRNA, Empty Vector, Flag-AUF1 p37, p40, p42, or p45 as well as WI-38 cells were collected at PDL 15 and 55 and also transfected with Control siRNA, AUF1 siRNA, HuR siRNA. For Ribo-Seq HeLa cells were transfected with Control siRNA, AUF1 siRNA, or HuR siRNA.

2014-11-04 | E-GEOD-52973 | biostudies-arrayexpress

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