HEK and HeLa cells: transfection of siRNA directed against GFP versus mock transfection
ABSTRACT: We tested the most widely used control siRNA directed against GFP for off-target effects and found by genome-wide expression profiling that it deregulates in addition to GFP a set of endogenous target genes. The detected modulated mRNA had target sequences homologous to the siRNA as small as 9 bp in size. However, we found no restriction of sequence homology to 3'UTR of target genes. Keywords: RNAi knock down Overall design: Two-condition experiment, siGFP transfected versus mock transfection, one replicate per array, one colour-switch per array
Project description:KEAP1 overexpressed and NRF2 siRNA knockdown A549 NSCLC cells were used to identify downstream genes of NRF2 pathway separately and by combinatorial analysis. We used triplicate microarrays of transfected A549 cells with mKeap1-GFP for overexpression, siRNAs targeting NRF2 for knockdown and siGFP as control respectively. As a result, we identified several genes which are involved in cancer metabolic functions in these cells. We used microarrays to identify the gene downregulated in both KEAP1 overexpressed and NRF2 siRNA knockdown A549 NSCLC cells and found a subset of downregulated genes which are involved in metabolic functions. Overall design: We divided microarrays into three groups. One group with A549 cells that were stably transfected with mKeap1-GFP construct for overexpression, second group with knockdown of NRF2 with specific siRNA and third group contains siGFP transfected control cells. Triplicates of both KEAP1 overexpression and NRF2 siRNA knockdown groups were analyzed and compared with control microarray data.
Project description:HCT116 cells were transfected with two different siRNA's targeting either DDX5, an siRNA targeting EBNA1, or no siRNA (mock). The siRNA targeting EBNA1 is used as a negative control since HCT116 cells do not have the EBNA1 gene. RNA was obtained from cultures at 24hrs post-siRNA transfection using the Qiagen RNeasy Minikit (cat. # 74104) with on-column DNase digestion performed as per the manufacturer's protocol. The RNA samples were isolated at 24hrs post-siRNA transfection since this timepoint precedes an impaired G1-to-S phase cell cycle progression phenotype that is evident at 48hrs post-siRNA transfection and so may reveal gene expression changes occuring before this effect on cell cycle. RNA samples were submitted to the Cold Spring Harbor Laboratory Microarray Faciity where cDNA was prepared, labeled, and hybridized to Affymetrix GeneChip Human Gene 1.0 ST microarrays. Data from the arrays were processed using the RMA method with an up-to-data probe set definition (Biostatistics 4:249-264 and Nucleic Acids Research 33(20):e175. Gene set analysis was performed using generally applicable gene set enrichment (BMC Bioinformatics 10:161). The most differentially regulated gene ontology groups were selected with FDR q-value < 0.1. Expression data from 3 independent HCT116 cutures transfected with either DDX5si2008, DDX5si2053, or EBNA1si1666 and 2 independent cultures transfected with mock conditions (no siRNA) were obtained from RNA samples prepared 24hrs post-siRNA transfection. These cultures were seeded with 200,000 cells per well 24hrs prior to siRNA transfection and were grown in DMEM media supplemented with 10% fetal bovone serum. Transfections were performed using lipofectamine RNAiMAX (Invitrogen 13778-075) and the manufacturer's protocol.
Project description:PAX3-FOXO1 is a fusion transcription factor characteristic for the majority of alveolar rhabdomyosarcoma tumors. It is the main oncogenic driver and deregulates expression of PAX3 target genes. The PAX3-FOXO1 target gene signature was determined in the Rh4 alveolar rhabdomyosarcoma cell line. Overall design: Rh4 cells were transfected with siRNA directed against PAX3-FOXO1 or scrambled control. RNA was isolated from these cells after 24, 48 and 72 h.
Project description:HOXA13 was depleted by siRNA mediated technology in Foreskin Fibroblast at 20nM and 100nM siRNA concentration. As a control siGFP was used at 20nM and 100nM for respective gene expression comparisons.
Project description:Purpose: Identifying target genes of the two human chromatin remodeling enzymes CHD3 and CHD4 Methods: see below in protocols Results: Libraries were sequenced on Illumina HiSeq2000 platform resulting in 37-71 Mio 50 bp paired-end reads per sample. We identified 16 (i) and 115 (ii) distinctly regulated genes when CHD3-GFP (i) or CHD4-GFP (ii) were overexpressed. Nine genes seem to be commonly regulated by CHD3 and CHD4. We successfully validated four genes from our RNA-seq via qPCR with two new (independent from those, used for RNA-seq) biological replicates. Conclusion: CHD3 and CHD4 regulate distinct genes. Overall design: Total RNA was prepared from 24 hours induced (1 ng/µl Dox) and non-induced Flp-In™ T-REx™ 293 cells, expressing GFP, hCHD3-GFP (UniProt: Q12873) or hCHD4-GFP(UniProt Q14839). Library preparation and Illumina Sequencing was perfprmed by EMBL GeneCore facility in Heidelberg (Germany: Dr. Vladimir Benes)
Project description:In order to understand the transcriptional effects of CD44s expression in a cell line that does not express CD44 in its native form we transfected CD44s into HEK cells and measured the transcriptional chances compared to native HEK cells Three biological replicates from each condition (native HEK cells and CD44s transfected cells) were measured using Affymetrix arrays
Project description:Analysis of differentially expressing genes in whole genome wide analysis of ALPPL2 expressing Panc-1 cells (Panc-1+ve) via siRNA mediated knockdown of ALPPL2 Panc-1 +ve and Panc-1-ve cell lines were generated from Panc-1 cell line based upon its hetergenous binding to aptamer SQ2. Detailed procedure of generation of these cell lines are described in Pooja Dua, Hye Suk Kang, Seung-Mo Hong, Ming-Sound Tsao, Soyoun Kim, and Dong-ki Lee. 2012 Alkaline Phosphatase ALPPL2 is a novel pancreatic carcinoma-associated protein. Cancer Research A four chip study using total RNA recovered from Panc-1+ve cells transfected with siALPPL2-2, siALPPL2-3, siGFP control and Lipofectamine 2000 treatament . Each chip measures 45,033 genes with three 60 mer probe pairs per target.
Project description:Peptidyl-prolyl isomerases (PPIs) are a superfamily of ubiquitous enzymes that catalyze the cis-trans interconversion of Xaa-Pro peptide bonds. The functions of most PPIs remain uncharacterized. FKBP25, a mammalian PPI, localizes to the nucleus, binds nucleic acids, and associates with chromatin modifying enzymes. However, the role of this protein in transcriptional regulation remains unclear. To help address this question we present RNA-seq gene expression profiling in HEK293 cells transfected with siRNA targeting FKBP25 relative to a GFP-targeting negative control. Overall design: Human embryonic kidney cells (HEK 293) were transfected with siRNA targeting FKBP25 or a GFP-targeting negative control and incubated for 72 h. Cells were harvested with trypsin, pelleted, and flash frozen before mRNA purification using the µMACs mRNA isolation kit (Miltenyl Biotec, Bergisch Gladbach, Germany). Purified mRNA was then analysed on an Agilent 2100 Bioanalyzer using Agilent 6000 RNA Nano Kit (Agilent Technologies, Santa Clara, California). cDNA was generated using the Superscript Double-Stranded cDNA Synthesis kit (ThermoFisher), 75bp paired-end libraries prepared using the Paired-End Sample Prep Kit (Illumina, San Diego, California) and then sequenced on a HiSeq 2000 sequencing system (Illumina).
Project description:We asked whether the human drosha protein, an RNase III homolog known to process microRNAs (miRNAs), might also be a small nuclear RNA (snRNA) 3' processing factor. Using retroviral siRNA silencing constructs, we stably knocked down drosha protein to nearly undetectable levels. Knockdown cells exhibited reduced growth rates and viability compared to controls, but no accumulation of unprocessed U2 snRNA precursors. In fungi, RNase III homologs process rRNA precursors and certain mRNAs. Although rRNA processing appears to be normal in the drosha knockdown cells, expression microarray analysis revealed misregulation of several mRNAs involved in cell growth and proliferation. Curiously, drosha knockdown appeared to downregulate the predicted mRNA targets of several miRNAs Experiment Overall Design: The experimental goal was to evaluate gene expression changes induced by siRNA knockdown of drosha. Four samples of HeLa cells were transfected with retroviral siRNA expression vectors: two replicates of an anti-GFP siRNA vector (siGFP) and two different anti-drosha siRNA vectors (sidroshaB and sidroshaC). Cells were selected with puromycin 24 hours after transfection and harvested 72 hours after transfection. Trizol-harvested RNA was processed with standard Affymetrix protocols and hybridized to U133Plus2.0 GeneChips. Signals were scaled to an arbitrary global mean value of 800.
Project description:The mechanism by which micro (mi)RNAs control their target gene expression is now well understood. It is however less clear how the level of miRNAs themselves is regulated. Under specific conditions, abundant and highly complementary target RNA can trigger miRNA degradation by a mechanism involving nucleotide addition and exonucleolytic degradation. One such mechanism has been previously observed to occur naturally during viral infection. To date, the molecular details of this phenomenon are not known. We report here that both the degree of complementarity and the ratio of miRNA/target abundance are crucial for the efficient decay of the small RNA. Using a proteomic approach based on the transfection of biotinylated antimiRNA oligonucleotides, we set to identify the factors involved in target-mediated miRNA degradation. Among the retrieved proteins, we identified members of the RNA-induced silencing complex, but also RNA modifying and degradation enzymes. We further validate and characterize the importance of one of these, the Perlman Syndrome 3’-5’ exonuclease DIS3L2. We show that this protein interacts with Argonaute 2 and functionally validate its role in target-directed miRNA degradation both by artificial targets and in the context of mouse cytomegalovirus infection. Overall design: To show the importance of TUT1 and DIS3L2 in target-induced miRNA decay we knocked-down by RNAi these two genes, either individually or in combination, and measured the effect of antimiRNA oligonucleotides by cloning and sequencing small RNAs. As a negative control, we used an siRNA directed against the RLUC gene. We generated one library per condition.