Absolute quantification of gene expression in biomaterials research using real-time PCR

Leong, David Tai; Gupta, Anurag; Bai, Hui Fen; Wan, Guoqiang; Yoong, Li Foong; Too, Heng‐Phon; Chew, Fook Tim; Hutmacher, Dietmar W.

doi:10.1016/j.biomaterials.2006.09.011

Cited by 73 publications

(55 citation statements)

References 18 publications

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“…Reverse transcription was conducted with ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan). Absolute quantitative real-time polymerase chain reaction (PCR) was performed using the method described by Leong et al (2007) with slight modifications. Primers were designed for preparation of UGT standard solution (Table 1) and synthesized by Greiner Bio-one (Tokyo, Japan).…”

Section: Methodsmentioning

confidence: 99%

Effects of Phenobarbital on Expression of UDP-Glucuronosyltransferase 1a6 and 1a7 in Rat Brain

Sakakibara

Katoh

Kondo

et al. 2015

Drug Metabolism and Disposition

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UDP-glucuronosyltransferase (UGT), a phase II drug-metabolizing enzyme, is expressed in the brain and can catalyze glucuronidation of endogenous and exogenous substrates in the brain. Thus, changes in UGT1A expression could affect homeostasis and drug efficacy. Phenobarbital (PB), a typical inducer of drug-metabolizing enzymes, has been reported to induce oxidative stress and epigenetic changes, which could alter UGT expression in the brain. Here, we aimed to clarify the effects of PB on Ugt1a6 and Ugt1a7 gene expression in rat brains. Sprague-Dawley rats were treated intraperitoneally with PB (80 mg/kg), once daily for 7 days. Ugt1a6 and Ugt1a7 mRNA expression levels were increased in the striatum and thalamus (Ugt1a6, 3.0-and 2.9-fold, respectively; Ugt1a7, 2.6-and 2.6-fold, respectively). Acetaminophen glucuronidation was also increased in the medulla oblongata and thalamus by 1.8-and 1.2-fold, respectively. The induction rates within different brain regions were correlated with Ugt1a6 and Ugt1a7 mRNA expression, and the degree of induction also correlated with that of NF-E2-related factor-2 mRNA. Measurement of oxidative stress markers suggested that PB induced oxidative stress in brain regions in which Ugt1a6 and Ugt1a7 mRNAs were increased. Moreover, histone modifications were altered by PB treatment, resulting in increased histone H3 lysine 4 trimethylation in the striatum and thalamus and decreased histone H3 lysine 9 trimethylation in the thalamus. These results suggested that oxidative stress and histone modifications may promote transcriptional activation of Ugt1a6 and Ugt1a7 genes. In summary, Ugt1a6 and Ugt1a7 mRNA levels were increased by PB treatment, which may alter pharmacokinetics in the brain.

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Section: Methodsmentioning

confidence: 99%

Effects of Phenobarbital on Expression of UDP-Glucuronosyltransferase 1a6 and 1a7 in Rat Brain

Sakakibara

Katoh

Kondo

et al. 2015

Drug Metabolism and Disposition

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“…Total RNA was prepared using TRIzol reagent (Invitrogen, USA), and cDNA was synthesized by PrimeScript ® RT reagent kit (Takara, China). HIF-1α and VEGF mRNA expression was evaluated quantitatively by real-time RT-PCR with SYBR ® Premix Ex Taq™ (Takara) and ABI PRISMR 7900HT real-time PCR system (35). The thermocycler conditions were preheating at 95˚C for 30 sec, cycles of 95˚C for 5 sec, 60˚C for 30 sec.…”

Section: Real-time Reverse Transcription Polymerase Chain Reaction (Rmentioning

confidence: 99%

Effects of trichostatin A on HIF-1α and VEGF expression in human tongue squamous cell carcinoma cells in vitro

Kang

Que

et al. 2012

Oncol Rep

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Abstract. Hypoxia is an essential feature of the microenvironment of solid tumors, which regulates a variety of transcription factors including hypoxia-inducible factor-1α (HIF-1α). HIF-1α overexpression enhances tumor angiogenesis via upregulation of vascular endothelial growth factor (VEGF) and some other hypoxia-inducible angiogenic factors, which lead to a more aggressive tumor phenotype, tumor metastasis and resistance to radiation and chemotherapy. In this study, we found that a histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), inhibited cell proliferation and invasion, blocked the cell cycle, and induced cell apoptosis in a dose-and time-dependent manner in the human tongue squamous cell carcinoma (TSCC) SCC-6 cell line in vitro. Furthermore, TSA reduced both basal levels and hypoxia-induced HIF-1α protein accumulation but not HIF-1α mRNA levels, and both protein and mRNA levels of VEGF expression. These results showed that TSA had a potent anticancer activity on TSCC cells, suggesting that TSA could be a promising drug targeting tumor angiogenesis via inhibition of HIF-1α and VEGF expression in the development of an effective chemopreventive and anticancer agent on human TSCCs.

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“…For absolute quantification of spliced/unspliced cib1 mRNAs, RT-PCR was performed on serial dilutions of calibrated solutions of 500-bp cib1 mRNA fragment with (unspliced) or without the intron (spliced) to generate calibration curves (Bio-Rad Icycler software). Absolute numbers of unspliced and spliced cib1 mRNA molecules from U. maydis strains grown in axenic culture or during pathogenic development were then calculated by plotting the respective CT values of the RT-PCR analysis against the calibration curves ( Leong et al, 2007). Calibration samples were processed in triplicates and all other samples in duplicates.…”

Section: Strains and Growth Conditionsmentioning

confidence: 99%

TheUstilago maydisClp1 Protein Orchestrates Pheromone andb-Dependent Signaling Pathways to Coordinate the Cell Cycle and Pathogenic Development

et al. 2010

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Regulation of the cell cycle and morphogenetic switching during pathogenic and sexual development in Ustilago maydis is orchestrated by a concerted action of the a and b mating-type loci. Activation of either mating-type locus triggers the G2 cell cycle arrest that is a prerequisite for the formation of the infectious dikaryon; this cell cycle arrest is released only after penetration of the host plant. Here, we show that bW, one of the two homeodomain transcription factors encoded by the b mating-type locus, and the zinc-finger transcription factor Rbf1, a master regulator for pathogenic development, interact with Clp1 (clampless 1), a protein required for the distribution of nuclei during cell division of the dikaryon. In addition, we identify Cib1, a previously undiscovered bZIP transcription factor required for pathogenic development, as a Clp1-interacting protein. Clp1 interaction with bW blocks b-dependent functions, such as the b-dependent G2 cell cycle arrest and dimorphic switching. The interaction of Clp1 with Rbf1 results in the repression of the a-dependent pheromone pathway, conjugation tube formation, and the a-induced G2 cell cycle arrest. The concerted interaction of Clp1 with Rbf1 and bW coordinates a-and b-dependent cell cycle control and ensures cell cycle release and progression at the onset of biotrophic development.

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Absolute quantification of gene expression in biomaterials research using real-time PCR

Cited by 73 publications

References 18 publications

Effects of Phenobarbital on Expression of UDP-Glucuronosyltransferase 1a6 and 1a7 in Rat Brain

Effects of Phenobarbital on Expression of UDP-Glucuronosyltransferase 1a6 and 1a7 in Rat Brain

Effects of trichostatin A on HIF-1α and VEGF expression in human tongue squamous cell carcinoma cells in vitro

TheUstilago maydisClp1 Protein Orchestrates Pheromone andb-Dependent Signaling Pathways to Coordinate the Cell Cycle and Pathogenic Development

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