PCR array screening for gene expression changes in HCC model comparing sorafenib treated or control samples
ABSTRACT: Candidate genes were successfully screened by Cancer PathwayFinder PCR Array Cancer PathwayFinder PCR Array was applied to compare gene changes in xenograft tumor samples of HCC model. A total of 12 samples were examined with 6 from Sorafenib treated group and 6 from control.
Project description:Purification of mouse mRNAs encoding signal transducer and activator of transcription 3 (Stat3) identified many miRNAs using multiplex miRNA array in RAW264.7 cells. To investigate the miRNAs targeting to mouse Stat3 gene in macrophage cells, we performed a protocol miRIP to affinity purify the miRNAs from an endogenous segment of Stat3 mRNA using nucleic acid hybridization and use multiplex miRNA array to identify the associated miRNAs.
Project description:MicroRNAs (miRNAs), small non-coding RNAs that fine-tune gene expression, play multiple roles in the cell, including cell fate specification. We have analyzed the differential expression of miRNAs during fibroblast reprogramming into induced pluripotent stem cells (iPSCs) and endoderm induction in iPSCs upon treatment with high concentrations of Activin-A in reduced serum. During reprogramming, adult mouse fibroblasts are converted into cells that resemble embryonic stem cells (ESCs) according to standard molecular and functional assays for pluripotency. The reprogrammed iPSCs assume an ESC-like miRNA signature, marked by the strong induction of pluripotency clusters miR-290-295 and miR-302/367 and conversely the downregulation of the let-7 family. On the other hand, endoderm induction in iPSCs results in the upregulation of 13 miRNAs. Given that the liver and the pancreas are common derivatives of the endoderm, the comparison of the expression levels of these 13 upregulated miRNAs with those in hepatocytes and pancreatic islets suggests a trend of miRNA upregulation in the endoderm tending towards an islet phenotype rather than that of a hepatocyte. These observations provide insights into how differentiation may be guided more efficiently towards the endoderm and further into the liver or pancreas. Moreover, we also report novel miRNAs enriched for each of the cell types analyzed. Stemloop RT-qPCR gene expression profiling. REPROGRAMMING: Differentially expressed miRNAs were determined between iPSCs (n=5 clones) and parent tail-tip fibroblasts (n=5) using mESCs R1 (n=3) and D3 (n=3). DIFFERENTIATION: Differentially expressed miRNAs were also analyzed in two iPSC clones upon treatment with Activin-A (n=2 each), and between primary mouse hepatocytes (n=3) and pancreatic islets (n=3).
Project description:Wild type (WT), Nod2-/-, Pglyrp3-/-, and Pglyrp3-/-Nod2-/- mice (BALB/c) were treated with oral 5% dextran sodium sulfate (DSS) for 48, 72, or 96 hrs, their colons were removed and homogenized, and RNA was isolated using the TRIZOL method. Quantitative reverse transcription real-time PCR (qRT-PCR) was used to quantify the amounts of mRNA in the colon using the inflammatory gene expression RT2 Profiler PCR Array from Qiagen/SA Biosciences. qRT-PCR gene expression profiling
Project description:Wild type (WT) and Pglyrp1-/- mice were treated with PBS or sensitized 5 days/week for 3 or 5 weeks with 10 µl per application of 2.5 mg/ml of purified house dust mite allergen. 3 days after the last sensitization the lungs were removed and homogenized, and RNA was isolated from the right lobes using the TRIZOL method. Quantitative reverse transcription real-time PCR (qRT-PCR) was used to quantify the amounts of mRNA in the lungs using custom RT2 Profiler PCR Arrays designed by us and manufactured by Qiagen/SA Biosciences. qRT-PCR gene expression profiling
Project description:Epithelial-mesenchymal transition (EMT) has recently been recognized as a key element of cell invasion, migration, metastasis, and drug resistance in several types of cancer, including non-small cell lung cancer (NSCLC). Our aim was to clarify microRNA (miRNA) -related mechanisms underlying EMT followed by acquired resistance to epidermal growth factor receptor tyrosine-kinase inhibitor (EGFR-TKI) in NSCLC. MiRNA expression profiles were examined before and after transforming growth factor-beta1 (TGF-β1) exposure in four human adenocarcinoma cell lines with or without EMT. Correlation between expressions of EMT-related miRNAs and resistance to EGFR-TKI gefitinib was evaluated. MiRNA array and quantitative RT-PCR revealed that TGF-β1 significantly induced overexpression of miR-134, miR-487b, and miR-655, which belong to the same cluster located on chromosome 14q32, in lung adenocarcinoma cells with EMT. MAGI2 (membrane-associated guanylate kinase, WW and PDZ domain-containing protein 2), a predicted target of these miRNAs and a scaffold protein required for PTEN (phosphatase and tensin homolog), was diminished in A549 cells with EMT after the TGF-β1 stimulation. Overexpression of miR-134 and miR-487b promoted the EMT phenomenon and affected the drug resistance to gefitinib, whereas knockdown of these miRNAs inhibited the EMT process and reversed TGF-β1-induced resistance to gefitinib. Our study demonstrated that the miR-134/487b/655 cluster contributed to the TGF-β1-induced EMT phenomenon and affected the resistance to gefitinib by directly targeting MAGI2, whose suppression subsequently caused loss of PTEN stability in lung cancer cells. The miR-134/miR-487b/miR-655 cluster may be new therapeutic targets in advanced lung adenocarcinoma patients, depending on the EMT phenomenon. miRNA expression profiles before and after TGF-β1 exposure were assessed in the four lung adenocarcinoma cell lines, A549, LC2/ad, PC3, and, PC9 by TaqMan miRNA arrays. Relative ratios of miRNAs in cells after TGF-β1 exposure were calculated when compared with the cells before TGF-β1 exposure.
Project description:Adult human ependymal and ventral horn regions were obtained from postmortem frozen samples by Laser Capture Microdissection. Briefly, Cryostat 25 micron sections from were stained with toluidin blue and both regions microdissected and collected on eppendorf (n=4 for each region). Samples mRNA concentration and purity was assessed by electrophoresis (BioRad Experion HighSensitivity kit, USA). RQI values were lower than 6,5 in every case, so that purification was followed by 2 cycle amplification with a kit designed for highly degraded samples (ExpressArt® TRinucleotide mRNA Amplification Kit; #6299-A15, AmpTec, AMSBIO, UK). After amplification, mRNA concentration and purity was assessed both by electrophoresis (BioRad Experion StSens kit, USA) and by spectrophotometry (Nanodrop, Thermo Scientific, USA). We amplified 3.7-37 ng of total RNA, obtaining between 6 and 21 µg of mRNA after 2 rounds. After collecting samples and studying the RNA integrity and quantity, cDNA of samples was selected for gene expression assays using 384 wells Custom Taqman Low Density Arrays. We built arrays with genes belonging to a profile of stemness or ependymoma (see Garcia-Ovejero et al., 2015, BRAIN). Taqman based qPCR gene expression profiling. Ependymal and ventral horn regions obtained by LCMD from four different individuals each were used to establish genes involved in stem cell niches or in ependymoma phenotype that are enriched in control human ependyma using ventral horn as a non-ependymary, non-neurogenic region. Samples were treated as stated in the summary. Equal amount of amplified RNA (aRNA; 25ng, corresponding approximately to 500ng total RNA) from each donor was used in Custom Designed Taqman Low Density Arrays. Every value is the resultant of duplicates at least, but most of them have been assayed 4 times.
Project description:In order to identify new markers of vascular cell senescence with potential in vivo implications, primary cultured Human Umbilical Vein Endothelial Cells (HUVECs), were analysed for microRNA (miR) expression. QRT-PCR microRNA expression profiling in 3 senescent (XIII passage) vs. 3 young HUVECs (II passage).
Project description:Inflammatory arthritis is associated with bone loss and fractures due to abnormal bone remodelling. Bone remodelling is 'uncoupled' with bone resorption increased and bone formation suppressed. These changes resemble those seen in patients treated with therapeutic glucocorticoids, and in both of these situations, altered wnt signalling is implicated. Recent studies have highlighted the importance of the synovial fibroblast in mediating abnormal bone remodelling during inflammation. The wnt antagonist dickkopf-1 (DKK1) is secreted by synovial fibroblasts in response to inflammation, and this protein has been proposed to be a master regulator of bone remodelling in inflammatory arthritis. Here we show that DKK1 expression by primary human synovial fibroblasts is more potently regulated by glucocorticoids than pro-inflammatory cytokines. Glucocorticoids, but not TNF-alpha, regulated expression of multiple wnt agonists and antagonists in favour of inhibition of wnt signalling. In vitro TNF-alpha and IL1-beta indirectly regulate DKK1 production through increased expression of the glucocorticoid activating enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1). These results demonstrate that the links between synovial inflammation, altered wnt signalling and bone remodelling may not be direct but are dependent on local activation of endogenous glucocorticoids. Human fibroblast-like synoviocytes isolated from patients with rheumatoid arthritis treated with either vehicle, TNF or dexamethasone (dex). Gene arrays for control, TNF and dexamethasone treatments were performed on three separate synovial fibroblast cell lines isolated from three rheumatoid arthritis patients. All fold changes displayed are the combined results of the three separate fibroblast lines.
Project description:A commercially available gene array (SABiosciences array PAMM-029ZD-12) was used to assay gene expression in skeletal muscle of SMA mice and control littermates. The Taiwanese severe SMA mouse model was used (Hsieh-Li et al. 2000). The array included 84 genes associated with DNA damage detection, DNA repair, apoptosis, cell cycle, and other functions. SMA group (n=3) vs. control group (n=3) - single technical replicates, triple biological replicates
Project description:we report a novel nanomedicine (Gd@C82(OH)22 ) effectively inhibit human breast tumor growth by antiangiogenesis in vivo. To further identify which angiogenic factor(s) were affected on mRNA level, the "RT² Profiler™ PCR Array Mouse Angiogenesis (APMM-024, SuperArray Bioscience Corporation)" was used. Keywords: nanomedicine, Gd@C82(OH)22, angiogenesis, MCF-7, breast cancer In the experimental group, the tumor bearing mice were administered intraperitoneally (i.p.) [Gd@C82(OH)22]n saline solution once a day at the dose of 2.5mM/kg, after the tumor tissue implantation into the animal for 7 days, continuing until the mice were sacrificed. The 0.9% saline soultion was used as control. At the end of the experiment, parts of the tumor tissue was collected, and further be analyzed by PCR-array.