Gene expression data at 24hrs post-siRNA transfection for HCT116 cultures transfected with either DDX5si2008, DDX5si2053, or EBNA1si1666 siRNA's or mock transfected.
ABSTRACT: 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:This transcriptome study is intended to discover the effects of MYC and TP63 on human keratinocytes differentiation at the genome level. Human keratinocyte cell line, HaCaT cells were transfected by siRNAs with the help of transfection reagent, INTERFERin. 48 hours post-transfection, total RNA was extracted from cells. We used a non-targeting siRNA as a negative control. siRNA targeting MYC or TP63 was obtained from QIAGEN, and RNAs from wild type HaCaT cells were used as reference samples. We did 3 biological replicates and 2 'dye swap' for each sample. Totally we got 12 samples, with 2 dye-swap for each sample, and 48 arrays used for all hybridization.
Project description:The transcription factors PAX3 and MITF are required for the development of the neural crest and melanocyte lineage, and both proteins play important roles in melanoma cell growth and survival. PAX3 transcriptionally activates MITF expression during neural crest development, but the relationship between these transcription factors during melanocyte development and in melanoma cells is currently poorly understood. This study aimed to further our understanding of the interaction between transcriptional networks controlled by PAX3 and MITF by assessing the effect of siRNA-mediated knockdown of PAX3 and MITF in metastatic melanoma cell lines. The goals of this study were to determine (i) if PAX3 is required for maintaining expression of MITF in melanoma and melanocyte cell lines; (ii) whether PAX3 and MITF independently, or redundantly, influence growth and survival in melanoma cell lines; and (iii) to investigate the respective roles of PAX3 and MITF expression in melanoma cell differentiation. Microarrays were used to measure global changes in transcript expression in response to siRNA-mediated knockdown of PAX3 or MITF compared to non-targeting controls in two metastatic melanoma cells lines. RNA was isolated from two different metastatic melanoma cell lines 30 hours after one of four different treaments: (i) transfection with siRNA targeting PAX3; or (ii) transfection with siRNA targeting MITF; or (iii) or transfection with siRNA targeting luciferase (non-targeting negative control); or (iv) treatment with media only (control). Therefore, eight samples were used for gene expression profiling by using GeneChip arrays, with one replicate per cell line per treatment.
Project description:Potential vorinostat-resistance candidate genes were identified using RNA interference screening in vorinostat-resistant HCT116 cells (HCT116-VR) using a synthetic lethal approach. In order to understand the mechanisms by which these genes contributed to vorinostat response, transcriptomic analysis was conducted on HCT116-VR cells and those with siRNA-mediated knockdown of each of the vorinostat resistance candidate genes. There are 45 samples in total, from triplicate independent biological experiments of 15 samples each. The negative control to which all gene knockdowns are compared is the mock transfection control (mock).
Project description:SH-SY5Y cells were transfected with an siRNA against T-UC.300A (final concentration 50nM) or negative control siRNA (Dharmacon Negative Control #1, final concentration 50 nM) using the transfection reagent Lipofectamine (Invitrogen). Media was changed after 24hrs. RNA was extracted 120hrs after transfection. Gene expression microarray analysis was carried out using Roche NimbleGens 4x72K Homo Sapiens gene expression array.
Project description:The objective of this study was to evaluate the consequences of Ago1 knockdown in metastatic prostate cancer cells PC3. To this end we profiled RNA from PC3 cells 72 hours following the transfection with a scramble siRNA (siGL3) and a siRNA targeting Ago1 (siAgo1). We profiled RNA from PC3 cells 72 hours following the transfection with a scramble siRNA (siGL3) and a siRNA targeting Ago1 (siAgo1). siAgo1 vs siGL3 gene expression profiling, two technical replicates with dye swap labeling scheme.
Project description:The ETS transcription factor GABPA controls cell migration in breast epithelial cells through targeting a cohort of genes, independently from another family member ELK1, and thereby achieves biological specificity. Here, both direct and indirect target genes for GABPA are identified by siRNA silencing of GABPA. Therefore, although ELK1 and GABPA ultimately control the same biological process, they do so by regulating distinct cohorts of target genes associated with actin cytoskeletal functions and cell migration control.
Project description:In order to investigate the role of CD34 antigen in haematopoietic commitment, we silenced the CD34 gene expression in CD34+ stem/progenitor cells using a siRNA approach. Experiment Overall Design: To maximize siRNA transfection efficiency, we utilized the NucleofectorTM technology (Amaxa). CD34+ cells were transfected with a mixture of 4 siRNAs targeting CD34 mRNA and with a non-targeting siRNA as a negative control. The expression level of CD34 antigen on control cells (MOCK and negative control treated cells) and CD34siRNA treated cells was assessed by immunofluorescence analysis at 24 and 48h post-nucleofection.
Project description:To investigate the function of CITED1 in melanoma, its expression was transiently down regulated using CITED1-targeting siRNA. The HT144 melanoma cell line was chosen as it had a relatively high level of detectable CITED1 mRNA and protein expression. HT144 melanoma cells were transfected with 2 siRNAs targeting CITED1 (4 replicates each) and a negative control non-targeting siRNA (4 replicates). RNA was harvested at 48 h post-transfection.
Project description:MDA231, BT549, and SUM159PT basal-like breast cancer cell lines were transfected with non-targeting siRNA (siCONTROL), siRNA targeting DUSP4 (siDUSP4), or siCONTROL + 4 or 24 hr of 1uM selumetinib. Cells were harvested at 96 hr post-siRNA transfection. Data were Log2 RMA normalized. We sought to identify changes in gene expression after MEK inhibition, or after loss of DUSP4 function in breast cancer cell lines.