Peripheral regions and central regions from HT-29 tumors
ABSTRACT: Gene expression of peripheral regions and central regions from HT-29 tumors Sample tissues were immediately frozen by liquid nitrogen after isolation. Total RNAs were extracted from samples with a PureLink RNA Mini Kit (Life Technologies). The integrity of total RNAs was evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Low Input Quick Amp Labeling Kit, one-color (Agilent Technologies) was used to prepare Cy3-labelled target cRNA according to the manufacturer's instructions. Labeled cRNAs were hybridized with a SurePrint G3 Human GE 8×60K Microarrays (Agilent Technologies). Three separate hybridizations were performed for each sample. Array images were captured using a DNA Microarray Scanner (Agilent Technologies), and data were analyzed using Feature Extraction Software (Agilent Technologies) to obtain background-corrected signal intensities. Data were further analyzed with GeneSpring GX Software (Version 11.0, Agilent Technologies). After data filtering, mRNAs differentially expressed in target cells versus controls were assessed by Fisher’s exact test, followed by multiple corrections using the Benjamini and Hochberg false discovery rate (FDR) method. Gene sets with a FDR q-value < 0.05 were considered to be significant.
Project description:LincRNA HOTAIR was expressed in pure SCLC and higher expression was significantly related to lymphatic invasion and relapse. Multivariate analyses demonstrated that HOTAIR expression correlated with RFS. In vitro experiments demonstrated that half of SCLC cell lines expressed HOTAIR at higher levels than normal cells and that, using cells of SBC-3, knockdown of HOTAIR decreased proliferative activity and cellular invasiveness with altered expression of cell adhesion-related genes. Total RNAs from one siHOTAIR-transfected SBC-3 cells and control cells (siGFP-transfected cells), were extracted using RNeasy mini kit Plus (Qiagen) and hybridized to the microarrays, Sure Print G3 Human GE 8x60K microarrays (Agilent Technologies, Santa Clara, CA, USA), according to the manufacturer’s instructions. Subsequently, data analysis was carried out using the GeneSpring GX 12 software (Agilent Technologies), with a stringency of P<0.1 and a 2-fold or more change using gene ontology analysis.
Project description:Wild-derived mice have contributed to mouse genetics by their genetic diversity that may increase the chance of identifying novel modifier genes responsible for specific phenotypes and diseases. However, gene-targeting using wild-derived mice has been unsuccessful due to the unavailability of stable embryonic stem cells. Here, we report that the CRISPR/Cas9-mediated gene-targeting can be applied to the Japanese wild-derived MSM/Ms strain. We targeted the nonagouti (a) gene encoding the agouti protein localized in hairs and the brain. We obtained three homozygous knockout mice as founders, all showing black coat color. While homozygous knockout offspring were physiologically indistinguishable from wild-type littermates, they showed specific domesticated behaviors; a high locomotion during the light period and a decline in the avoidance of a human hand. These phenotypes were consistent over the subsequent generations. Our findings support the empirical hypothesis that nonagouti is a domestication gene, which might repress aggressive behavior. Global gene expression patterns in the midbrain of wildtype and nonagouti (a) homogygous mutant MSM/Ms mice were analyzed by one-color Mouse Gene Expression 8x60K microarray. The midbrain tissues were manually isolated from 8-9 weeks old mice under dissecting microscope. Total RNA was purified from dissected midbrain using Trizol (Thermo Fisher Scientific). Purified total RNA was amplified and labeled with Cy3 using Low-Input QuickAmp Labeling Kit (Agilent Technologies). Cy3-labeled RNAs were hybridized to SurePrint G3 Mouse Gene Expression v2 8x60K Microarray Kit (Agilent Technologies) at 65 °C for 17h. After wash, the hybridized slides were scanned with DNA microarray scanner (Agilent Technologies). The scanned images were processed with Feature Extraction software (Agilent Technologies) to extract signal intensities of each probe. The extracted signal data were imported into the Gene Spring GX 13.1.1 software (Agilent Technologies) and normalized using the default settings. Two and six biological replicates were performed in wildtype and mutant group, respectively.
Project description:We examined FASN knockdown LNCaP cells obtained by shRNA transduction with Mission lentiviral transduction particles (SHCLNV-NM 00410, TRCN3128, Sigma) (FASN-RNAi cells). In this study, we used cells transfected with non-targeting shRNA as a control (control-RNAi cells). The expression of genes related to cellular proliferation (phospholipase A2, group IVA, PLA2G4A; tensin 3, TNS3; glypican 4 GPC4), cell adhesion and extracellular matrix organization [peroxidasin homolog (Drosophila) PXDN; sarcoglycan epsilon, SGCE; von Willebrand factor, VWF; hydroxysteroid (17-beta) dehydrogenase 12, HSD17B12; cysteine-rich secretory protein LCCL domain containing 2, CRISPLD2], and cell motility (TNS3, RAP2B member of RAS oncogene family, RAP2B) were shown to be down-regulated by FASN inhibition with RNAi. FASN inhibition led to down-regulation of the PLA2G4A and HSD17B12 genes encoding phospholipase A2 and 17-beta hydroxysteroid dehydrogenase, respectively, which are the key enzymes related to production of an intracellular second messenger arachidonic acid and androgen hormones, both playing roles in promotion of tumor progression. We also found that the genes related to arachidonic acid signalling, including RGS2, SPAG16, VWF and RAP2B, were also suppressed with FASN inhibition. Gene expression profiling therefore demonstrated that FASN inhibition induces down-regulation of genes related to cell proliferation, cell adhesion, migration, and invasion, as well as the production of arachidonic acid and androgen hormones, both of which drive tumor progression. Total RNA isolation was performed with a Micro-to-Midi total RNA purification system (Invitrogen). The integrity of total RNAs was evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies). Low Input Quick Amp Labeling Kit, one-color (Agilent Technologies) was used to prepare Cy3-labelled target cRNA according to the manufacturer's instructions. Labeled cRNAs were hybridized with a SurePrint G3 Human GE 8×60K Microarrays (Agilent Technologies). Two separate hybridizations were performed for each sample. Array images were captured using a DNA Microarray Scanner (Agilent Technologies), and data were analyzed using Feature Extraction Software (Agilent Technologies) to obtain background-corrected signal intensities. The data were further analysed with GeneSpring GX Software (Version 11.0, Agilent Technologies). After filtering of data, mRNAs differentially expressed in target versus control were considered using the Fisher exact test, followed by multiple corrections using the Benjamini and Hochberg false discovery rate (FDR) method. Gene sets with a FDR q-value < 0.05 were considered significant.
Project description:To further development of our gene expression profile of Alzheimer's disease, we have employed whole genome microarray expression profiling as a discovery platform to identify genes with the potential biomarker for early disease detection Total RNA from peripheral blood cells was extracted, reverse-transcribed and labelled, then analysed for gene expression using GeneSpring GX12 (Agilent Technologies, USA). About 180 samples (AD=90, Non-demented control=90) was used for microarray analysis.
Project description:To identify genes hypermethylated and transcriptionally downregulated, we have employed the Illumina Infinium HumanMethylation450 BeadChip and Agilent SurePrint G3 Human Gene Expression 8x60K.And we validated selected genes in cases and controls. eventually we estalished CIMP of MDS. The gene expressions of four cases and four controls were measured.
Project description:Comparison of gene expression in pterygium fibroblast cells after 48 h treatment with siRNA to knock down expression of linc-9432 or non-specific oligonucleotide control 21 nM pooled siRNA (Ambion, Life Technologies) or non-specific control oligonucleotides are added to cultured fibroblast cells for 48 hours
Project description:To clarify downstream genes regulated by TSHZ2, MCF-7 cells were lentivirally transduced by TSHZ2 and its control LacZ and served their RNA samples for gene expression analysis using AGILENT human 8x60K cDNA microarray. Array analysis of MCF-7 cells expressing either TSHZ2 or LacZ. 1 color method was employed.
Project description:To clarify transcriptional target genes of GLI1 in human mammary epithelial cells and breast cancer cells, primary culture cells of human mammary epithelium HMEC and breast cancer cell line MCF-7 were lentivirally transduced by either GLI1 or its control LacZ. Their RNA samples were served for expression analysis using AGILENT human 8x60K cDNA microarray. Array analysis of HMEC and MCF-7 expressing either GLI1 or LacZ. 1 color method was employed.
Project description:Neural stem cells reside in a hypoxic microenvironment within the brain. However, the crucial transcription factors that regulate neural stem cell biology under physiologic hypoxia are poorly understood. Here, we have performed microarray analysis of hypoxic versus normoxic neural stem cells with the aim of identifying pathways and transcription factors that are activated under oxygen concentrations mimicking normal brain tissue microenvironment. To identify the molecular mechanisms mediating the effect of low oxygen levels on NSC biology, we profiled the global effect of sustained, physiologic hypoxia on NSC gene expression using an unbiased approach by genome-wide microarray analysis. NSC were isolated from E13.5 mouse embryos (OF-1 strain) as previously described (Johe et al., 1996). After isolation, cells were immediately cultured at 37°C, 5% CO2 and either 5% or atmospheric (≈21%) oxygen and maintained in these conditions for 2-3 passages (minimum of 10 days). Then, total RNA was extracted, labeled and hybridized in a SurePrint G3 Mouse GE 8x60k array (Agilent Technologies). Four independent experiments were performed using different mouse donors for each experiment.
Project description:In Arabidopsis thaliana, cytokinin responsive B-type ARR transcription factors and HD-ZIP III transcription factors such as REVOLUTA (REV), act cooperatively as master regulators of shoot regeneration. To identify the downstream targets of ARR-HD-ZIP III transcriptional complex, we used an inducible line of REV, 35S::FLAG-GR-rREV, in which FLAG-tagged miR165/6-non-targetable form of REV (rREV)-GR fusion protein was expressed from 35S promoter. DEX treatment induced activation of REV by translocation of FLAG-GR-rREV fusion protein from cytoplasm to the nucleus. We treated 35S::FLAG-GR-rREV seedlings with 6-benzylaminopurine (6-BA, a cytokinin), dexamethasone (DEX), or 6-BA+DEX for 2 hours. Total RNAs were extracted and subjected to Agilent Arabidopsis Gene Expression Microarray analyses. The differentially expressed genes (>1.5-fold, p<0.05) were identified. 10-day-old 35S::FLAG-GR-rREV plants were treated with 6-benzylaminopurine (6-BA), dexamethasone (DEX), or 6-BA+DEX for 2 hours. DEX treatment induced activation of REV by translocation of FLAG-GR-rREV fusion protein from cytoplasm to the nucleus. Total RNA was extracted with RNeasy Mini Kit and hybridized to Agilent Arabidopsis Gene Expression Microarray. Differentially expressed genes were defined by a 1.5-fold expression difference with a P value<0.05. Biological replicates were performed.