PCR array screening for gene expression changes in HCC model comparing sorafenib treated or control samples
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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: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.
Project description:Global miRNA expression profiling of human malignancies is gaining popularity in both basic and clinically driven research. But to date, the majority of such analyses have used microarrays and quantitative real-time PCR. With the introduction of digital count technologies, such as next-generation sequencing (NGS) and the NanoString nCounter System, we have at our disposal, many more options. To make effective use of these different platforms, the strengths and pitfalls of several miRNA profiling technologies were assessed, including a microarray platform, NGS technologies and the NanoString nCounter System. These results were compared to gold-standard quantitative real-time PCR. Comparison of non-small cell lung cancer cell lines grown in vitro (n = 5) and in vivo (n = 5) as xenograft models.
Project description:The monohaloacetic acids (monoHAAs) are generated as byproducts during the disinfection of drinking water and are cytotoxic, genotoxic, mutagenic, and teratogenic. Iodoacetic acid (IAA) toxicity was mitigated by antioxidants, suggesting the involvement of oxidative stress. Other monoHAAs may share a similar mode of action. Human oxidative stress and antioxidant defense gene arrays (SA biosciences) were used to evaluate changes in transcriptome profiles in the human intestinal epithelial cell line FHS 74 INT generated by three compounds, chloroacetic acid (CAA), bromoacetic acid (BAA) and IAA at two time points (30 min and 4 h). Twelve samples were evaluated. Each treated sample was paired with a concurrent negative control (cells treated in medium only). Three technical repeats were included for each sample and Ct values were calculated from the average of the three repeats. Samples 1,2,and 3 were isolated from 30 min negative controls for CAA, BAA, and IAA respectively. Samples 4, 5, and 6 were isolated from cells treated for 30 min with CAA, BAA, and IAA respectivley. Samples 7, 8, and 9 were isolated from 4h negative controls for CAA, BAA, and IAA respectively. Samples 10, 11, and 12 were isolated from cells treated for 4 h with CAA, BAA and IAA respectively. Ct values were normalized against the average of the 5 housekeeping genes included in the array to generate M-NM-^TCt values. Fold changes for each gene were calculated as a ratio of 2^-M-NM-^TCttest / 2^-M-NM-^TCtcontrol.
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-M-NM-21) 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-M-NM-21 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-M-NM-21 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-M-NM-21-induced resistance to gefitinib. Our study demonstrated that the miR-134/487b/655 cluster contributed to the TGF-M-NM-21-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-M-NM-21 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-M-NM-21 exposure were calculated when compared with the cells before TGF-M-NM-21 exposure.
Project description:Wild type (WT) and Pglyrp1-/- mice were treated with PBS or sensitized 5 days/week for 3 or 5 weeks with 10 M-BM-5l 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: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:MicroRNAs (miRNAs) are important in the regulation of many biological processes including muscle development. However, little is known regarding miRNA regulation of muscle regeneration. In mature murine tibialis anterior muscle following injury, 298 miRNAs were significantly changed during the time course of muscle regeneration including 86 that were altered greater than 10-fold as compared to uninjured muscle. Temporal miRNA expression patterns were identified and included inflammation-related miRNAs (miR-223 and -147) that increased immediately after injury; this pattern contrasted to that of mature muscle-specific miRNAs (miR-1, -133a and -499) that were abruptly decreased following injury and then up-regulated in later regenerative events. Another cluster of miRNAs were transiently increased in the early days of muscle regeneration. This included miR-351, a miRNA that was also transiently expressed during myogenic progenitor cell (MPC) differentiation in vitro. Based on computational predictions, further studies demonstrated that E2f3 was a target of miR-351 in myoblasts. Moreover, knockdown of miR-351 expression inhibited MPC proliferation and promoted apoptosis during MPC differentiation, whereas miR-351 overexpression protected MPC from apoptosis during differentiation. Collectively, these observations suggest that miR-351 is involved in both the maintenance of MPC proliferation and the transition of MPC into differentiated myotubes. Thus, a novel, time-dependent sequence of molecular events during skeletal muscle regeneration has been identified, i.e., miR-351 inhibits E2f3 expression, a key regulator of cell cycle progression and proliferation, and promotes MPC proliferation and protects early differentiating MPC from apoptosis, important events in the hostile tissue environment after acute muscle injury. Skeletal muscles are damaged and repaired repeatedly throughout life. Muscle regeneration maintains locomotor function during aging and delays the appearance of clinical symptoms in neuromuscular diseases, such as Duchenne muscular dystrophy. The capacity for skeletal muscle growth and regeneration is conferred by satellite cells located between the basal lamina and the sarcolemma of mature myofibers. Upon injury, satellite cells reenter the cell cycle, proliferate, and then exit the cell cycle either to renew the quiescent satellite cell pool or to differentiate into mature myofibers. Despite recent advances, genes involved in these processes are still largely unknown. Understanding the molecular mechanisms that regulate satellite cell activities could promote development of novel countermeasures to enhance muscle regeneration that is compromised by diseases or aging. Using a muscle injury mouse model, we profiled miRNA expression during muscle regeneration.
Project description:Compared mRNA expression between control and insulin treated cells. HT29 cells were treated with 100nM Insulin for 24h. The RT2 Profiler PCR Arrays PAHS-30C (SA Biosciences, MD, USA) was designed to analyze 84 genes related to human insulin signaling pathway. The RT-PCR was carried out using an ABI PRISMM-BM-. 7000 Sequence Detection System (Applied Biosystems, Foster City, CA).The expression of HSD11B2 for the 3 experiments concerned was monitored in parallel by real time PCR which confirming significant downregulation by insulin. qPCR gene expression profiling. RNA from 3 separated experiments was collected.
Project description:Purpose: To identify tissue microRNAs predictive of sunitinib activity in patients with metastatic renal-cell carcinoma (MRCC) and to validate them in a cellular model. Selected microRNAs were studied in serum from MRCC patients and healthy individuals. Methods: We screened 673 microRNAs using TaqMan Low-density Arrays (TLDAs) in tumors from MRCC patients with extreme phenotypes of marked efficacy and resistance to sunitinib, selected from an identification cohort (n=41). Differentially expressed microRNAs were selected using bioinformatics-based target prediction analysis and quantified by qRT-PCR in tumors from patients presenting similar phenoytpes selected from an independent cohort (n=117). Results were validated in a cellular model of sunitinib resistance and studied in serum from healthy individuals and MRCC patients. Results: TLDAs identified 64 microRNAs differentially expressed in the identification cohort. Seven candidates were quantified by qRT-PCR in the independent series. MiR-942 was the most accurate predictor of sunitinib efficacy (p=0.0074). High expression of miR-942, miR-133a, miR484, and miR-628-5p was significantly associated with decreased time-to-progression and overall survival. These microRNAs were overexpressed in the sunitinib resistant cell line Caki-2 in comparison with the sensitive parental cell line. Serum levels of miR-942, miR-133a, miR-484, miR-146a-5p, miR-374a and miR-486-5p were significantly reduced in MRCC patients compared to healthy controls. Conclusions: Our strategy identified differentially expressed microRNAs in MRCC patients presenting marked sensitivity and resistance to sunitinib. Mir-942 was the best predictor of efficacy. Results were confirmed in a cellular model of sunitinib resistance. We also identified exosome derived serum microRNAs differentially expressed in MRCC patients and healthy individuals. Taqman Low Density Array for 6 FFPE tissues obtained from extreme phenotype MRCC patients, (n=3 marked resistance to sunitinib treatment patients and n=3 marked sensitivity to sunitinib treatment patients), was performanced to screen 667 microRNAs.