A Quantitative Method of Determining Initial Amounts of DNA by Polymerase Chain Reaction Cycle Titration Using Digital Imaging and a Novel DNA Stain

Becker, Andreas; Reith, Annette; Napiwotzki, Jörg; Kadenbach, Bernhard

doi:10.1006/abio.1996.0230

Cited by 55 publications

(26 citation statements)

References 2 publications

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“…Total RNA was treated with DNase I before reverse transcription to ensure removal of all contaminating DNA. PCR primers were designed with PrimerExpress software developed by Applied Biosystems (Foster City, CA) for optimal product length, GC content, and melting temperature for quantitative reverse transcription-PCR (RT-PCR) using SYBR Green I DNA binding dye technology (18). Primers span at least one intron whenever possible to ensure exclusive amplification of cDNA.…”

Section: Methodsmentioning

confidence: 99%

The G Protein–Coupled Receptor S1P2 Regulates Rho/Rho Kinase Pathway to Inhibit Tumor Cell Migration

et al. 2005

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Sphingosine 1-phosphate (S1P) is a lysophospholipid that exerts a variety of responses in cells such as proliferation, migration, and survival. These effects are mediated by G protein-coupled receptors on the cell surface (S1P 1-5 ), which activate downstream signaling intermediates such as Rac and Rho GTPases. Mechanisms of S1P action in human glioblastoma cells are not well defined. S1P receptors (1-5) and S1P-metabolizing enzymes were expressed in three human glioblastoma cell lines. S1P had a profound and differential effect on glioblastoma cell migration. U87 cells treated with S1P showed a significant increase in migration, whereas U118 and U138 cell lines were strongly inhibited. S1P-mediated inhibition correlated with S1P 2 receptor expression. FTY720-P, an S1P analogue that binds all S1P receptors except S1P 2 , did not inhibit glioblastoma cell migration. Overexpression of S1P 2 further suppressed migration, and blockage of S1P 2 mRNA expression by small interfering RNA reversed the inhibitory effect. Contrary to previous reports showing bimodal regulation of Rac activity and migration by S1P 2 receptor stimulation, both Rac1 and RhoA GTPases were activated by S1P treatment in native cells and cells overexpressing S1P 2 . Treatment of U118 cells with the Rhoassociated protein kinase (ROCK) inhibitor Y-27632 restored migration suggesting that ROCK-dependent mechanisms are important. Actin staining of S1P stimulated U118 cells overexpressing B-galactosidase resulted in pronounced stress fiber formation that was exacerbated by S1P 2 overexpression, partially blocked by S1P 1 , or totally abolished by pretreatment with Y-27632. These data provide evidence of a novel mechanism of S1P inhibition of tumor cell migration via Rho kinase-dependent pathway. (Cancer Res 2005; 65(9): 3788-95)

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Section: Methodsmentioning

confidence: 99%

The G Protein–Coupled Receptor S1P2 Regulates Rho/Rho Kinase Pathway to Inhibit Tumor Cell Migration

et al. 2005

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“…After the images were recorded in a computer, the band intensities were processed with the NIH Image program (version 1.62) as described previously. 25 These band intensities were used to calculate the ratio of human-specific marker (hGAPDH, hBNP, hcTn-I, hMHC-␣, hMHC-␤, hNkx 2.5, hSMA, hsm22␣, hKDR, and hCD31) expression to total GAPDH expression.…”

Section: Rt-pcr Analysis Of Cd34 ؉ Cells and Ischemic Heart Tissuementioning

confidence: 99%

Dose-Dependent Contribution of CD34-Positive Cell Transplantation to Concurrent Vasculogenesis and Cardiomyogenesis for Functional Regenerative Recovery After Myocardial Infarction

et al. 2006

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Background— Multilineage developmental capacity of the CD34 + cells, especially into cardiomyocytes and smooth muscle cells (SMCs), is still controversial. In the present study we performed a series of experiments to prove our hypothesis that vasculogenesis and cardiomyogenesis after myocardial infarction (MI) may be dose-dependently enhanced after CD34 + cell transplantation. Methods and Results— Peripheral blood CD34 + cells were isolated from total mononuclear cells of patients with limb ischemia by apheresis after 5-day administration of granulocyte colony-stimulating factor. PBS and 1×10 3 (low), 1×10 5 (mid), or 5×10 5 (high) CD34 + cells were intramyocardially transplanted after ligation of the left anterior descending coronary artery of nude rats. Functional assessments with the use of echocardiography and a microtip conductance catheter at day 28 revealed dose-dependent preservation of left ventricular function by CD34 + cell transplantation. Necropsy examination disclosed dose-dependent augmentation of capillary density and dose-dependent inhibition of left ventricular fibrosis. Immunohistochemistry for human-specific brain natriuretic peptide demonstrated that human cardiomyocytes were dose-dependently observed in ischemic myocardium at day 28 (high, 2480±149; mid, 1860±141; low, 423±9; PBS, 0±0/mm 2 ; P <0.05 for high versus mid and mid versus low). Immunostaining for smooth muscle actin and human leukocyte antigen or Ulex europaeus lectin type 1 also revealed dose-dependent vasculogenesis by endothelial cell and SMC development after CD34 + cell transplantation. Reverse transcriptase–polymerase chain reaction indicated that human-specific gene expression of cardiomyocyte (brain natriuretic peptide, cardiac troponin-I, myosin heavy chain, and Nkx 2.5), SMC (smooth muscle actin and sm22α), and endothelial cell (CD31 and KDR) markers were dose-dependently augmented in MI tissue. Conclusions— Human CD34 + cell transplantation may have significant and dose-dependent potential for vasculogenesis and cardiomyogenesis with functional recovery from MI.

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“…After 24 h of infection, RNA was purified from these cultures and used to generate cDNA. We employed a technique for quantitative RT-PCR analysis, in which a fluorescent dye (SYBR Green) that binds double-stranded DNA is used to quantitate the amount of amplicon as it accumulates during the PCR reaction (22,(35)(36)(37). For each cDNA sample, we measured the cycle number at which PCR product accumulation reaches a defined threshold.…”

Section: Activation Of Endogenous Egr1 Target Genes-previousmentioning

confidence: 99%

EGR1 Target Genes in Prostate Carcinoma Cells Identified by Microarray Analysis

Svaren¹,

Ehrig²,

Abdulkadir³

et al. 2000

Journal of Biological Chemistry

166

133

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The EGR1 transactivator is overexpressed in prostate cancer, and its expression pattern suggests that EGR1 could potentially regulate a number of steps involved in initiation and progression of prostate cancer, such as mitogenesis, invasiveness, angiogenesis, and metastasis. To identify potential EGR1 target genes in an unbiased manner, we have utilized adenovirus-mediated expression of EGR1 in a prostate cancer cell line to identify specific genes that are induced by EGR1. Using oligonucleotide arrays, a number of EGR1-regulated genes were identified and their regulation was confirmed by quantitative reverse transcription-polymerase chain reaction analysis. One of the largest gene classes identified in this screen includes several neuroendocrine-associated genes (neuron-specific enolase, neurogranin), suggesting that EGR1 overexpression may contribute to the neuroendocrine differentiation that often accompanies prostate cancer progression. This screen also identified several growth factors such as insulin-like growth factor-II, platelet-derived growth factor-A, and transforming growth factor-␤1, which have previously been implicated in enhancing tumor progression. The insulin-like growth factor-II gene lies within the 11p15.5 chromosomal locus, which contains a number of other imprinted genes, and EGR1 expression was found to induce at least two other genes in this locus (IPL, p57 KIP2 ). Based on our results, coupling adenoviral overexpression with microarray and quantitative reverse transcription-polymerase chain reaction analyses could be a versatile strategy for identifying target genes of transactivators.

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A Quantitative Method of Determining Initial Amounts of DNA by Polymerase Chain Reaction Cycle Titration Using Digital Imaging and a Novel DNA Stain

Cited by 55 publications

References 2 publications

The G Protein–Coupled Receptor S1P2 Regulates Rho/Rho Kinase Pathway to Inhibit Tumor Cell Migration

The G Protein–Coupled Receptor S1P2 Regulates Rho/Rho Kinase Pathway to Inhibit Tumor Cell Migration

Dose-Dependent Contribution of CD34-Positive Cell Transplantation to Concurrent Vasculogenesis and Cardiomyogenesis for Functional Regenerative Recovery After Myocardial Infarction

EGR1 Target Genes in Prostate Carcinoma Cells Identified by Microarray Analysis

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