Gene expression analysis of liver and colon cancer primary tumors and metastasis
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE40367 | GEO | 2015/01/22
SECONDARY ACCESSION(S): PRJNA173769
REPOSITORIES: GEO
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Project description:To identify molecular changes underlying the chemopreventive or tumor promoting effects of SRD5A inhibition, we profiled gene expression changes in benign prostate epithelium from patients with PCa treated with dutasteride, a dual SRD5A inhibitor. Subjects were aged 45-80 years with clinically localized PCa (T1C to T2b), Gleason score <7, and serum PSA 2.5-10 ng/dL. 81 men were randomized to immediate RP (n=25) or to dutasterdie at 0.5 mg (n=26) or 3.5 mg (n=24) orally per day for four months prior to RP. Prostate samples embedded in OCT were used for laser capture microdissection (LCM). Keywords: clinical trial, Dutasteride, PEDB, dose response, prostate cancer, microarray Subjects were aged 45-80 years with clinically localized PCa (T1C to T2b), Gleason score <7, and serum PSA 2.5-10 ng/dL. 81 men were randomized to immediate RP (n=25) or to dutasterdie at 0.5 mg (n=26) or 3.5 mg (n=24) orally per day for four months prior to RP. Prostate samples embedded in OCT were used for laser capture microdissection (LCM). Approximately 2000-3000 epithelial cells per sample were collected from 8µm sections using the Arcturus Veritas™ Laser Capture Microdissection System according to the Arcturus HistoGene LCM Frozen Section Staining Kit Protocol (Mountain View, CA). Total RNA was isolated using the Arcturus Picopure™ Kit, and the samples were treated with DNAse using the Qiagen RNase-Free DNase Set (Qiagen Inc, Valencia, CA). Total RNA was subjected to two rounds of linear amplification using the Ambion MessageAmpII Kit (Ambion Inc, Austin, TX), quantitated in a Gene-Spec III spectrophotometer (Hitachi, Tokyo) and aRNA integrity evaluated using gel electrophoresis. 40 Human Prostate (PEDB) Microarrays were run with sample on the Cy3 channel against a Human Gold Standard (v5) on the Cy5 channel.
2008-12-01 | E-GEOD-9972 | biostudies-arrayexpress
Project description:Indomethacin (an inhibitor of prostaglandin synthesis) and RU486 (mifepristone, progesterone receptor antagonist) are well known compounds which impair follicle rupture in rats, nevertheless the histopahotlogical figures of these follicle were different in detail due to the difference of the pathway which each compound inhibits. To characterize these unruptured follicles further and to investigate the genes which were altered in each rat treated with indomethacin or mifepristone, gene expression profile in ovarian follicle were analyzed in female sprague-Dawley rats. Peri- or post ovulatory follicles were collected by laser microdissection and extracted RNA was analyzed by genechip after single dosage of mifepristone or indomethacin. Animals were singly administered orally Indomethacin (IM) at 4mg/kg at 16:00 or RU486 (RU) at 100 mg/kg at 10:00 on the proestrus day, and sacrificed by exsanguination under anesthesia at 22:00 on the proestrus day (PeF: peri-ovulatory follicle), and 10:00 on the estrus day (PoF: post-ovulatory follicle). Ovaries were also collected from untreated rats at 22:00 on the proestrus day, and 10:00 on the estrus day as control groups. Three animals were contained per each treatment group at each timing. The ovaries were removed and embedded in OCT (Tissue-Tek, Sakura Finetek, Torrance, CA), frozen in dry-ice-cold isopentan, and stored at -70 C until used. Frozen sections (8 micro meter thick) were mounted on the membrane slides (MMI, Glatteburg, Zurich, Switzerland), and stained with HistogeneTM LCM frozen section staining kit (Arcturus Engineering, Mountain View, CA). Then, 15 follicles in each phase were retrieved by Laser Micro Dissection system (LMD, CellCut Plus, MMI). The collected follicle sections were lysed by the buffer RLT in the collection tube supplied in RNeasy Micro Kit (Qiagen, Hilden, Germany), and total RNA was extracted according to manufacturer's instructions.
2014-02-01 | E-GEOD-54584 | biostudies-arrayexpress
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2008-06-13 | E-GEOD-5018 | biostudies-arrayexpress
Project description:Spatially resolved, single-cell transcriptome analysis of high quality remains challenging, despite the rise of high-resolution spatial transcriptomics. Laser-capture microdissection (LCM) is widely used to isolate cells of interest from tissue sections. Here, we developed DRaqL (Direct RNA recovery and quenching for LCM), a technique that allows efficient lysis of single, LCM-isolated cells from alcohol- and formalin-fixed tissue and stained frozen sections, and that is amenable to enzymatic reactions for cDNA amplification within the same sampling tubes. Quantitative evaluations showed that single-cell RNA sequencing combined with DRaqL allowed transcriptomic profiling from frozen sections at an efficiency comparable with that from freshly dissociated cells, with small biases and errors in lowly expressed genes, in addition to allowing exon-exon junction profiling. By applying this method to mouse ovarian frozen sections, we revealed a transcriptomic continuum of growing oocytes quantitatively associated with the size of oocytes and follicles, identified genes highly correlated with oocyte diameters, and detected oocyte-specific splice isoforms. We further identified genes that were differentially expressed in granulosa cells depending on their distance from oocytes, suggesting distinct epigenetic regulatory mechanisms and cellular proliferation. Thus, DRaqL provides an efficient cell lysis for single-cell cDNA amplification from frozen sections that is amenable to high-quality RNA sequencing analysis.
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Project description:Entamoeba histolytica contains a large and novel family of transmembrane kinases (TMK) with proposed roles that include both amebic response to the environment and immune evasion. In the later case, the process requires several elements --these include a large gene family encoding antigenically distinct surface proteins and the expression of one variant antigen at a time by a single pathogen. Laser-capture microdissection was used in conjunction with microarray analysis to demonstrate that single trophozoites express more than one TMK gene. Overall, our data indicates that multiple members of the novel E. histolytica TMK family are utilized for non-redundant functions by the parasite. For laser capture microdissection analysis, harvested trophozoites were allowed to adhere to metal framed PEN membrane slides (Molecular Devices, Sunnyvale, CA) for 15 mins at 37°C in TYI-S-33 media. Subsequently, single cells were captured from the PEN slides using a Pix Cell II laser capture microdissection system equipped with an Olympus microscope (Arcturus Engineering, Mountain View, CA).
2010-11-01 | E-GEOD-19064 | biostudies-arrayexpress
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2006-04-07 | GSE4381 | GEO
Project description:Topical treatment with N-nitrosotris-(2-chloroethyl)urea (NTCU) induces lung squamous cell carcinoma in mouse. The aim of this study is to analyze the expression profile of the mouse NTCU-induced SCC compared to normal lung to identify gene expression changes in the SCC cells. NIH Swiss mice presenting lung SCC and control healthy mice were used for laser capture microdissection (LCM). LCM was performed in a LMD7000 microscope (Leica Microsystems GmbH, Wetzlar, Germany). RNA was extracted using the RNeasy FFPE kit (Qiagen, Hilden, Germany) and libraries were prepared from using the SMARTer Stranded Total RNA-Seq Kit – Pico Input Mammalian (Clontech Laboratories, Inc., Mountain View, CA, USA). Sequencing was performed using an Illumina HiSeq 2500 generating 50 base pair paired end reads. Reads of low quality were filtered, and adapters trimmed using Cutadapt v1.9.1. Alignment to the reference genome (UCSC mm10) was performed using TopHat v2.0.1, and RNA counts were generated for each gene using the Python package HTSeq v0.6.1, employing the “intersection-strict” overlap mode. DESeq2 was used to normalize and transform the data, examine the relationship between samples (by plotting the first two principal components) and to test for differential expression
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Project description:We have previously detected cyclin D1-initiated and p53-related (PERP; p53 apoptosis-associated target) cell death in a striatal 6-hydroxydopamine (6-OHDA)-treated Parkinsonian mouse model by adaptor-tagged competitive PCR (ATAC-PCR) (Synapse 51, 279-286, 2004). In order to establish the mechanism of dopaminergic cell death in Parkinsonâs disease, various Parkinsonian animal models should be studied. Therefore, the time-course alteration of gene expression in the substantia nigra pars compacta was investigated after a single administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in common marmosets. In the MPTP model, gene expression of neither cyclin D1 nor PERP was altered. There were also few common alterations in gene expression between the MPTP and 6-OHDA models. This suggests that the mechanism of cell death may differ between these models, and extensive study in various Parkinsonian models should be performed to elucidate the mechanisms of dopaminergic cell death in Parkinsonâs disease. Experiment Overall Design: Materials and methods Experiment Overall Design: 1. Animals Experiment Overall Design: Five 3-year-old male common marmosets (Callithrix jacchus) weighing 350â400 g were used. A single marmoset was used as a vehicle-treated control, with the rest being administered MPTP. A microarray experiment and an immunohistochemical study were performed in the same animals. They were colonized in the Center for Life Science Resources in Kagoshima University, Japan. Animals were housed with free access to standard food in an air-conditioned room under a constant 12 h light/dark cycle (lights on at 7:00 a.m.) at a temperature of 25â27°C and 45â55% humidity. All efforts were made to minimize animal suffering, and to reduce the number of animals used. The present experiments were carried out after obtaining permission from the Committee of Animal Experimentation, Kagoshima University Graduate School of Medical and Dental Sciences, Japan. Experiment Overall Design: 2. MPTP administration Experiment Overall Design: A single dose of MPTP was administered intravenously (i.v.) to the animals in order to examine the time-course alteration in gene expression. As in our previous study, the dose of MPTP was 2.5 mg/kg (Nomoto et al., 1997). At this dose, animals exhibit immobility during an entire day, while doses greater than 2.5 mg/kg can induce death. MPTP hydrochloride (Sigma, St. Louis, MO, USA) was dissolved in 0.9% saline, and administered under intraperitoneal pentobarbital anesthesia (40 mg/kg). Under deep anesthesia with pentobarbital, the animals were decapitated either at 2 h, 6 h, 1 day or 14 days after MPTP administration. All experiments using MPTP were performed in accordance with safety guidelines (Przedborski et al., 2001). Experiment Overall Design: 3. Laser capture microdissection Experiment Overall Design: The midbrain of each marmoset was dissected on an ice-cold glass plate, and mounted on a specimen block with O.C.T. compound (Tissue-Tek, Sakura Finetechnical, Tokyo, Japan). The midbrains were frozen by covering them with very finely powdered dry ice. Sections (8 μm thickness) were cut on a cryostat (Microm HM500S, Carl Zeiss Japan, Tokyo, Japan), and mounted on ice-cold, membrane-coated glass slides (Carl Zeiss Japan). The membranes on the glass slides were coated with poly-L-lysine (P8920; Sigma). Immediately after mounting, slides were cooled on dry ice and stored at â80°C for less than 3 days. Slides were fixed in ethanol/acetate solution (19:1) at 4°C for 3 min, and washed with PBS at 4°C for 1 min. Slides were stained with 0.1% toluidine blue at room temperature for 10 s, washed with PBS at 4°C for 10 s, and then air-dried for 5 min. Immediately after staining, slides were processed for laser capture microdissection (LCM). Experiment Overall Design: A Leica AS LMD (Leica Microsystems, Wetzlar, Germany) was used for LCM. SNc sections corresponded to level A4.5 of the brain atlas (Stephan et al., 1980). These sections contained transverse sections of the oculomotor nerve. A strip of the unilateral SNc was dissected (Fig. 1). The SNcs from two slides were collected and placed in a PCR tube containing 10 μl extraction buffer of the PicoPure RNA Isolation Kit (Arcturus Bioscience, Mountain View, CA, USA). Experiment Overall Design: 4. Microarray experiments Experiment Overall Design: Total RNA was extracted by the PicoPure RNA Isolation Kit, and eluted in approximately 10 μl buffer, according to the manufacturerâs protocol. In order to obtain a sufficient amount of amplified RNA (aRNA) for the microarray experiments, total RNA was amplified using two rounds of in vitro transcription. The first round of amplification was performed using the RiboAmp HS RNA Amplification Kit (Arcturus). The second round of amplification used the Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies, Palo Alto, CA, USA) and cyanine-3 or cyanine-5 labeling. The labeled aRNA was hybridized with Agilent whole human genome oligo microarray G4112A. In order to correct the buffer composition in the second round of amplification using the Low RNA Input Fluorescent Linear Amplification Kit, cDNA (11 μl) that was purified in the round 2 procedure by the RiboAmp HS RNA Amplification Kit, was added to the modified transcription buffer of the Agilent kit. Each sample contained 4 μl of 5 ï´ first strand buffer, 20 μl of 4 ï´ transcription buffer, 6 μl 0.1 M DTT, 8 μl NTP mix, 6.4 μl 50% PEG, 0.5 μl RNase inhibitor (RNase Out; Invitrogen, Carlsbad, CA, USA), 0.6 µl inorganic pyrophosphatase, 0.8 μl T7 RNA polymerase, 2.4 μl cyanine (PerkinElmer Life Sciences, Boston, MA, USA; cyanine-3 for controls and cyanine-5 for MPTP-treated marmosets), and 20.3 μl DW. Labeled aRNA was synthesized according to the instructions for the Agilent kit. Experiment Overall Design: An Agilent 2100 bioanalyzer and an RNA 6000 Pico LabChip Kit were used to examine the length distribution of labeled aRNAs. A preliminary experiment using the protocol found in the appendices of the HistoGene LCM Immunofluorescence Staining Kit (Arcturus) was performed to determine the degree of degradation of total RNA that occurred during tissue staining.
2008-06-13 | E-GEOD-5176 | biostudies-arrayexpress
Project description:In our investigations of the molecular pathways of prostate tumorigenesis in Nkx3.1; Pten mutant mice using gene expression profiling, we now find that the AP-1 transcription factors, c-Jun and c-Fos, are significantly up-regulated during cancer progression. Forced expression of c-Fos and c-Jun in prostate cancer cells results in increased tumorigenicity, activation of Erk MAP kinase, and enhanced survival in the absence of androgens, which are hallmarks of disease progression. In humans, Jun and Fos proteins are significantly up-regulated during prostate cancer progression and significantly correlated with activation of Erk MAP kinase. Most notably, expression of Jun is associated with disease recurrence independent of other currently used prognostic indicators. These analyses reveal a hitherto unappreciated role for AP-1 transcription factors in prostate cancer progression vis-à -vis Erk MAP kinase signaling, as well as the identification of a novel marker of disease recurrence, namely c-Jun. Experiment Overall Design: Mouse prostate was collected from wild-type or the Nkx3.1; Pten compound mutant mice at the age of 8-16 months. One lobe of dosolateral prostate was snap-frozen in OCT and stored at -80ºC for laser capture microdissection (LCM). To obtain androgen-independent lesions, mice were castrated at 7 to 14 months of age. Mice were sacrificed for analysis at 8 to 16 months of age and one dosolateral prostatic lobe was snap-frozen in OCT and stored at -80ºC for LCM. Approximate 1000 Prostate epithelial cells were isolated from normal prostate, dysplasia, prostatic intraepithelial neoplasia (PIN) or cancer lesions using PixCell IIE LCM system (Arcturus), followed by RNA linear amplification and labeling using Small Sample Labeling Protocol VII (Affymetrix). Samples were labeled using a BioArray High Yield RNA transcript labeling kit (Enzo Life Scientific) and were hybridized to MOE430A GeneChips containing 22,690 well characterized mouse genes/ESTs (Affymetrix).
2008-10-21 | E-GEOD-11836 | biostudies-arrayexpress