The differentially expressed genes in SRSF3 siRNA-treated HCT116 cells
ABSTRACT: Several members of SRSF family play wide-ranging roles in the regulation of transcription and post-splicing processes as well as splice sites selection. Although the expression of SRSF3 was reported to be overexpressed in several cancers, the roles of SRSF3 in the cancer cells are almost unknown. We analyzed differentially expressed genes in SRSF3 siRNA-treated HCT116 cells and identified the specific pathways regulated by SRSF3. After treatment of HCT116 cells with SRSF3 (sample 02) or control (sample 01) siRNA, total RNA was extracted using RNeasy Mini Kit (Qiagen, Valencia, CA). The quality of the purified RNA and its applicability for microarray analysis were assessed by the Agilent 2100 Bioanalyzer using a RNA 6000 Nano Labchip kit (Agilent Technologies, Palo Alto, CA, USA). Total RNA (400 ng) was used for amplification, labeling and hybridization to a whole human genome oligoDNA microarray (4x44k; Agilent) according to the manufacture’s instructions.
Project description:SRSF3 is overexpressed in human invasive ovarian cancer and its overexpression is required for cancer cell growth and survival. To decipher the mechnisms behind the role of SRSF3 in ovarian cancer, we examined the gene expression and splicing in the ovarian cancer cell line that was engineered to express a doxycycline-induced SRSF3 siRNA, which was able to knockdown SRSF3 expression by 90% and induce apoptosis. Overall design: Total RNAs extracted from A2780/SRSF3si2, a subline of ovarian cancer cell line A2780, treated with or without doxycycline at 0.1ug/ml for three days were analyzed using Affymetrix GeneChip® Human Exon 1.0 ST Array
Project description:SRSF3 is overexpressed in human invasive ovarian cancer and its overexpression is required for cancer cell growth and survival. To decipher the mechnisms behind the role of SRSF3 in ovarian cancer, we examined the gene expression and splicing in the ovarian cancer cell line that was engineered to express a doxycycline-induced SRSF3 siRNA, which was able to knockdown SRSF3 expression by 90% and induce apoptosis. Total RNAs extracted from A2780/SRSF3si2, a subline of ovarian cancer cell line A2780, treated with or without doxycycline at 0.1ug/ml for three days were analyzed using Affymetrix GeneChip® Human Exon 1.0 ST Array
Project description:RNA seqeuncing was performed to identifiy changes in genes expression and alternative splicing following SRSF3 depletion in pluripotent stem cells. Overall design: Induced pluripotent stem cells (iPSCs) generated from reprogrammable conditional SRSF3 knockout (SRSF3-KO/OKSM) mouse embryonic fibroblasts (MEFs) were induced for 24h to deplete SRSF3 and RNA seqeuncing was performed.
Project description:We have employed whole genome microarray expression profiling as a discovery platform to screen potential target genes of miR-204-5p in colorectal cancer cell HCT116. HCT116 cells were seeded in 6-cm2 tissue culture plates and transfected with the miR-204-5p mimic or negative control (NC) using Lipofectamine 2000 (Invitrogen, USA). After propagation for 48 hours, total RNA was extracted using TRIzol reagent (Invitrogen, USA). Expression profiling was performed using an Agilent human whole genome oligo microarray chip (4×44K) (Agilent, USA) Expression profiling in HCT116 cells was measured at 48 hours after transfection with miR-204-5p or negative control. Experiments were performed using the two samples without repeat experiment.
Project description:Human transformer 2β (Tra2β) is a serine/arginine-rich (SR)-like protein splicing factor and is now implicated to play wide-ranging roles in gene expression as an RNA-binding protein. In this study, we identified a subset of Tra2β-associated mRNAs in HCT116 human colon cancer cells using RNA immunoprecipitation with an anti-Tra2β antibody and microarray analysis. Whole-cell extracts from HCT116 cells were incubated with protein-A Sepharose beads precoated with 3 μg anti-Tra2β antibody or control rabbit IgG for 2 h at 4°C. RNA in the immunoprecipitation (IP) materials was measured by human whole-genome microarray (Agilent Technology).
Project description:We have independently knocked down TRA2B and SRSF3 in HEK293 cells to identify the sum of alternative splicing changes that results from depletion of each factor. Each was compared to a control transfection of the hairpin vector alone. In this set, 9 total samples were analyzed. There were three biological replicates for each of three conditions: knockdown control, TRA2B knockdown and SRSF3 knockdown. Arrays were analyzed for gene expression using GeneBase and for alternative splicing using MADS+ and OmniViewer software.
Project description:RNA was isolated from and YTHDC1, SRSF1, SRSF3, SRSF7, SRSF9 and SRSF10 deficient human HeLa cells using the TRIzol (Invitrogen) reagent by following the company manual. For all samples the RNA integrity was checked using an Agilent Bioanalyzer 2100. All samples showed a RIN (RNA integrity number) of higher than 9. Approximately 2.5 µg of total RNA was then used for library preparation using a TruSeq™ RNA Sample Prep Kit v2 (Illumina, San Diego, CA, USA) according to the manufacturer’s protocol.The libraries were sequenced using HiSeq2000 (Illumina) in single-read or paired-read mode, creating reads with a length of 101 bp. Sequencing chemistry v2 (Illumina) was used and samples were multiplexed in two samples per lane. Examination of gene expressive levels in normal and YTHDC1, SRSF1, SRSF3, SRSF7, SRSF9 or SRSF10 deficient Human HeLa cells
Project description:Macrophages infected with S. aureus were subjected to gene expression profiling to undertake a complete understanding of the interaction induced gene expression changes in both, S.aureus and the RAW macrophages. Agilent one-color experiment, Agilent-021933 Genotypic designed Custom Staphylococcus aureus and Mus musculus 8x15k
Project description:The differential gene expression study was conducted in the Anti - HMGA2 siRNA treated Retinoblastoma cell (Y79). The transient transfection in Y79 cells were performed using Anti-HMGA2 siRNA . The concentration and the time of incubation for the transfection was optimised to increase the silencing efficiency and reduce the off -target effects. After 48 hours of transfection the cells were collected and the whole genome cDNA microarray was performed to interpret the differential gene expression regulated by HMGA2 in Y79 cells. The results of the microarray data has been validated. One-color experiment,Organism: Human , Custom Whole Genome Human 8x60k designed by Genotypic Technology Pvt. Ltd. (Agilent-027114), Labeling kit: Agilent Quick-Amp labeling Kit (p/n5190-0442)