Microvesicular mRNA profiling of SW480 human colorectal adenocarcinoma cells
ABSTRACT: We analyzed mRNA profile of SW480-derived microvesicles using microarray Keywords: Microvesicular mRNA profiling Overall design: The total RNA was extracted from purified microvesicles derived from SW480 by the single-step isolation method using TRIzol reagent (Invitrogen Corporation, Carlsbad, CA) according to the manufacturer’s protocol. For microarray hybridizations, we followed protocol described in the instruction manual of Illumina TotalPrep RNA Amplification kit (Ambion, Austin, TX). In brief, 640 ng of total RNA from microvesicles was used to synthesize single-stranded cDNA using T7-Oligo(dT) promoter primer and RNase H-facilitated second-strand cDNA, which was purified and served as a template in the subsequent in vitro transcription. The in vitro transcription was carried out in the presence of T7 RNA polymerase and a biotinylated NTP mixture for cRNA. There are four replicate hybridizations. Same amount (Each 1.5 μg) of cRNA was hybridized to arrays, and subsequently labeled with Cy3-streptavidin (Amersham Biosciences, Little Chalfont, UK) and scanned with a Bead Station (Illumina, San Diego, CA).
Project description:Analysis of SW480 cells following knockdown of Ezrin using RNAi. Ezrin is a protein that regulate the organization of cytoskeleton. Ezrin KD SW480 was used to study the role of ezrin in colon cancer. Keywords: ezrin, colon cancer, migration Overall design: Knockdown ezrin in SW480 cells and investigate the affection on cell migration and the downstream genes.
Project description:ChIP-on-chip tilling array comparing untreated human SW480 cells vs SW480 cells treated with 2mM H2O2 for 30min. Exposing cells to the reactive oxygen species, hydrogen peroxide, recruits DNA methyltransferase 1 (DNMT1) to damaged chromatin. DNMT1 becomes part of a complex(es) containing DNMT3B and members of Polycomb Repressive Complex 4. The goal was to determine the effect of hydrogen peroxide treatment on chromatin, including changes in histone modifications and binding patterns of chromatin-associated proteins. [Agilent-014706]: Two-condition: untreated SW480 cells (U) vs H2O2 treated SW480 cells (T). Five-mark: SIRT1, gamma-HA2X, DNMT1, DNMT3B, and EZH2. [Agilent-014707]: Two-condition: untreated SW480 cells (U) vs H2O2 treated SW480 cells (T). Five-mark: SIRT1, gamma-HA2X, DNMT1, DNMT3B, and EZH2. [Agilent-026595]: Two-condition: untreated SW480 cells (U) vs H2O2 treated SW480 cells (T). Five-mark: SIRT1, gamma-HA2X, DNMT1, DNMT3B, and H3. [Agilent-027811]: Two-condition: untreated SW480 cells (U) vs H2O2 treated SW480 cells (T). Four-mark: H3, 3MeK4H3, AcK16H4, and 3MeK27H3.
Project description:Expression profiles were generated for eight genes located on chromosome 13 following RNAi to enable the determination of gene specific LOF RNAi signatures in the colorectal cancer cell line SW480. Triplicate transfections of SW480 cells were performed using two different siRNA duplexes corresponding to each gene target in 6-well plates using the same protocol scaled 30-fold. RNA was purified 72 hrs after transfection. RNA was also purified from negative control siRNA-transfected cells (AllStar siNegative (siNEG), Qiagen). Reduction in target mRNA levels was confirmed by real-time PCR prior to the array analysis.
Project description:Deleted in breast cancer (DBC1; also known as CCAR2) is a coactivator for nuclear receptors (NRs) and other transcription factors as well as a negative regulator of epigenetic modifiers such as deacetylases SIRT1 and HDAC3. We performed genome-wide gene expression analysis in wild-type and DBC1 knockout SW480 cells to investigate global gene expression changes induced by DBC1 knockout. Total RNAs were isolated from SW480 wild-type and DBC1 knockout cells using the RNeasy mini kit (Qiagen). The integrity of RNA was analyzed using an Agilent 2100 Bioanalyzer. Three independent biological replicates were assayed for each sample. The microarrays were performed following the Affymetrix standard protocol.
Project description:To characterize the molecular origin of primary lymphomas of the central nervous system (PCNSL), 21 PCNSL of immunocompetent patients were investigated by microarray-based gene expression profiling. Comparison of the transcriptional profile of PCNSL with various normal and neoplastic B cell subsets demonstrated PCNSL (i) to display gene expression patterns most closely related to late germinal center B cells, (ii) to display a gene expression profile similar to systemic diffuse large B cell lymphomas (DLBCL), and (iii) to be in part assigned to the activated B cell-like (ABC) or the germinal center B cell-like (GCB) subtype of DLBCL. Keywords: PCNSL – DLBCL – gene expression profiling Overall design: Total RNA was extracted from tissue blocks containing > 80% tumor cells using the TRIzol reagent (Invitrogen, Carlsbad, CA) and purified using RNeasy Kit (Qiagen, Valencia, CA). Double-stranded cDNA (ds-cDNA) was generated from 5 μg of total RNA using the SuperScript Double Stranded cDNA Synthesis Kit (Invitrogen) and a poly-dT oligonucleotide that contains T7 RNA polymerase initiation site (Sigma-Proligo, St. Louis, MO). The ds-cDNA was used as template to generate biotinylated cRNA by in vitro transcription using MEGA-script T7 High Yield Transcription Kit (Ambion, Austin, TX), biotin-11-CTP, and biotin-11-UTP (Perkin Elmer, Altham, MA). The biotinylated cRNA was purified by RNAeasy Kit (Qiagen) and fragmented according to the Affymetrix protocol. 15 μg of fragmented cRNA was hybridized to HG-U95Av2 microarrays (Affymetrix, Santa Clara, CA). Gene expression values were determined by GCOS1.2 software (Affymetrix) using the global scaling option.
Project description:Investigation of whole genome gene expression level changes in a colorectal cancer cell line SW480 expressing FOXC2, compared to the pBabe control cells. Genes associated with metastasis regulated by FOXC2 in colorectal cancer were analysed. The role of FOXC2 in breast cancer metastasis are further described in Mani SA, Yang J et al. Mesenchyme Forkhead 1 (FOXC2) plays a key role in metastasis and is associated with aggressive basal-like breast cancers. PNAS 2007; 104: 10069-10074 . A six chip study using total RNA recovered from three separate cultures of SW480/pBabe and three separate cultures of SW480/FOXC2. Each chip measures the expression level of 45033 genes from SW480/pBabe or SW480/FOXC2.
Project description:cRNAs were hybridized with HumU133A oligo arrays (Affymetrix, Santa Clara, CA) which contain 22,283 probe sets. Preliminary studies indicated that Affymetrix Human GeneChips detected more than 60% of pig liver transcripts. All Affymetrix data were filtered for those genes with fluorescent intensity value of less than 100. Genes whose expressions in livers from the folate deficient and ethanol fed groups were either increased or decreased by 2-fold or more (p<0.05) compared to those in livers from the control group were considered significantly changed. GeneChip cRNA probes used in GeneChip expressions were obtained by following a protocol from the manufacturer. First strand cDNA was synthesized by reverse transcription of 8 μg total RNA with superscript II reverse transcriptase (Invitrogen, Carlsbad, CA) and T7-oligo (dT) 24 primer (Genset Oligos, Boulder, CO), followed by second strand cDNA synthesis. Biotin-labeled cRNA probes were generated by reverse transcription of double stranded cDNA using BioArray High yield RNA transcript labeling kit (ENZO Life Sciences, Inc., Farmingdale, NY). The cRNA probes were purified from unincorporated nucleotides by RNeasy mini column (QIAGEN, Valencia, CA), fragmented and hybridized overnight at 680 C to the Human GeneChip array (HG U133A, Affymetrix). Keywords: parallel sample
Project description:Heat shock protein 90 (HSP90) is a molecular chaperone required for the stability and function of many proteins. The chaperoning of oncoproteins by HSP90 enhances survival, growth and invasive potential of cancer cells. HSP90 inhibitors are promising new anticancer agents, in which the benzoquinone ansamycin 17-allylamino-17-demethoxygeldanamycin (17-AAG) is currently in clinical evaluation. However, the implications of acquired resistance to this class of drug remain largely unexplored. In the present study, we have generated isogenic human colonic cancer cell line SW480 that is resistant to 17-AAG by continued culturing in the compound. We used microarrays to detail the global programme of gene expression underlying the acquired resistance to 17-AAG in SW480 and SW480-R cell lines. Overall design: SW480 cells were incubated and treated with 17-AAG at an initial concentration of 1×IC50. The medium with the compound was replaced every 48 h to discard dead cells. When surviving cells grow to 80% confluence, cells were passaged and the 17-AAG concentration was then added as follows: 2×, 4×, 6× ……IC50 till undulated. The generated resistant cell line(SW480-R) was cultured without 17-AAG at least of 2 weeks for RNA extraction and hybridization on Affymetrix microarrays, and for other analysis. Paired parental cells in another flask were serially passaged without 17-AAG as an untreated control.
Project description:Methylation analysis of normal lymphocytes, HCT116, RKO and SW480 In vitro methylated DNA (IVD) generated from normal lymphocytes, HCT116, RKO and SW480 genomic DNA was subjected to sodium bisulfite treatment and the DNA was analyzed for CpG methylation using the Infinium Methylation Array.
Project description:Comparision of Mus musculus Control and Treated samples. Overall design: Low RNA Input Fluorescent Linear Amplification Kit (Agilent, Santa Clara, CA) was used for labeling. Briefly, both first and second strand cDNA were synthesized by incubating 500ng of total RNA with 1.2ul of oligo dT-T7 Promoter Primer in nuclease-free water at 65 ◦C for 10 min followed by incubation with 4.0ul of 5× First strand buffer, 2ul of 0.1M DTT, 1 ul of 10mM dNTP mix, 1ul of 200 U/ul MMLV-RT, and 0.5ul of 40U/ul RNaseOUT, at 40 ◦C for 2 hour. Immediately following cDNA synthesis, the reaction mixture was incubated with 2.4 ul of 10 mM Cyanine-3-CTP or 2.4 ul of 10 mM Cyanine-5-CTP (Perkin-Elmer, Boston, MA), 20ul of 4X Transcription buffer, 8 ul of NTP mixture, 6 ul of 0.1M DTT, 0.5 ul of RNaseOUT, 0.6ul of Inorganic pyrophosphatase, 0.8 ul of T7 RNA polymerase, and 15.3ul of nuclease-free water at 40 ◦C for 2 hour. Qiagen’s RNeasy mini spin columns were used for purifying amplified aRNA samples. The quantity and specific activity of cRNA was determined by using NanoDrop ND-1000 UV-VIS Spectrophotometer version3.2.1. Samples with specific activity >8 were used for hybridization. 825ng of each Cyanine 3 or Cyanine 5 labeled cRNA in a volume of 41.8ul were combined with 11ul of 10x Blocking agent and 2.2ul of 25x Fragmentation buffer (Agilent), and incubated at 60deg C for 30 minutes in dark. The fragmented cRNA were mixed with 55ul of 2x Hybridization Buffer (Agilent). About 110ul of the resulting mixture was applied to the Microarray and hybridized at 65degC for 17 hours in an Agilent Microarray Hybridization Chamber (SureHyb: G2534A) with Hybridization Oven. After hybridization, slides were washed with Agilent Gene expression Wash Buffer I for 1 minute at room temperature followed by a 1 min wash with Agilent Gene expression Wash Buffer II for 37C. Slides were finally rinsed with Acetonitrile for cleaning up and drying. Microarrays were scanned on an Agilent scanner (G2565AA) at 100% laser power, 30% PMT.Data extraction was carried out with Agilent Feature Extraction software (version 9.1), and normalization was done using linear per array algorithm according to the manufacturer’s protocol. The data was analysed by GeneSpring GX . The differential expression was considered if the Log 2 mean of at least -1 for the down regulated genes and +1 for the upregulated genes. We considered only the genes that were reproducible from all replicates.