Quantitative detection of reverse transcriptase-PCR products by means of a novel and sensitive DNA stain.

Schneeberger, Christian; Speiser, Paul; Kury, F; Zeillinger, Robert

doi:10.1101/gr.4.4.234

Cited by 189 publications

(107 citation statements)

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“…Quantitative RT-PCR-Quantitative RT-PCR (qRT-PCR) was performed with 1 g of total RNA using the Taqman model 7700 sequence detection system (PerkinElmer Life Sciences) as described earlier (27)(28)(29)(30). Primer sequences used are: FKHRL1fwd, 5Ј-tcaatcagaacttgctccacca-3Ј; FKHRL1rev, 5Ј-ggactcactcaagcccatgttg-3Ј; FKHRfwd, 5Ј-aacctggcattacagttggcc-3Ј; FKHRrev, 5Ј-aaatgcaggaggcatgactacgt-3Ј; AFXfwd, 5Ј-ttttctcactgtgccaattaggg-3Ј; AFX rev, 5Ј-tccaacagcattgctcatcttg-3Ј; TRAILfwd, 5Ј-GACCTGCGTGCTGATCGTG-3Ј; and TRAILrev, 5Ј-TGTCCTGCATCTGCTTCAGCT-3Ј.…”

Section: Methodsmentioning

confidence: 99%

FOXO Proteins Regulate Tumor Necrosis Factor-related Apoptosis Inducing Ligand Expression

Modur

Nagarajan

Evers

et al. 2002

Journal of Biological Chemistry

340

299

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Mutations in PTEN occur in 60 -80% of prostate cancers and lead to a constitutive activation of the phosphatidylinositol 3-kinase pathway and a resultant loss of activity of the FOXO family of forkhead transcription factors FKHRL1 and FKHR. To provide insight into the role of PTEN mutations in prostate cancer, we used microarrays to identify genes regulated by FKHRL1 and FKHR in LAPC4 prostate carcinoma cells. These studies revealed that adenoviral overexpression of FKHRL1 and FKHR in the LAPC4 prostate cancer cell line resulted in apoptosis and induced the expression of many genes that affect cellular proliferation or survival. The expression of one of these FOXO-regulated genes, TRAIL, a pro-apoptotic member of the tumor necrosis factor family, was decreased in human metastatic prostate tumors. The altered expression of TRAIL in these tumors correlated directly with decreased PTEN expression and the resultant loss of FKHRL1 and FKHR activity. Analysis of the effects of FOXO proteins on the TRAIL promoter localized the FKHRL1 responsive element of the TRAIL promoter to nucleotides ؊138 to ؊121 and demonstrated that TRAIL is a direct target of FKHRL1. These findings suggest that the decreased activity of FKHRL1 and FKHR in prostate cancers resulting from loss of PTEN leads to a decrease in TRAIL expression that may contribute to increased survival of the tumor cells.The forkhead transcription factors are DNA-binding proteins characterized by the presence of a conserved 110-amino acid winged helix DNA binding domain (1). They play important roles in embryogenesis, tumorigenesis, and maintenance of differentiation status. In Caenorhabditis elegans the forkhead transcription factor DAF-16 is under control of the insulin receptor/PI 3-kinase 1 pathway (2). The human orthologs of DAF-16 include FKHRL1, FKHR, and AFX and belong to the FOXO subfamily of forkhead transcription factors. The PI 3-kinase pathway, via activation of its downstream kinase Akt, phosphorylates each of the FOXO proteins at three different Ser/Thr residues (3). These phosphorylated FOXO proteins interact with 14-3-3 proteins and are subsequently sequestered in the cytoplasm where they are inactive. Inhibition of the PI 3-kinase pathway by PTEN overexpression or pharmacologic means leads to dephosphorylation and nuclear translocation of active FKHRL1, FKHR, and AFX, which in turn leads to cell cycle arrest and apoptosis (4). Conversely, loss of PTEN activity results in increased Akt activity leading to inhibition of FOXO protein activity through their phosphorylation and cytoplasmic sequestration. In addition, the expression of dominant negative FKHRL1 results in the inhibition of apoptosis, demonstrating that FOXO transcriptional activity controls cellular proliferation and apoptosis downstream of PTEN (5).The PTEN gene was initially identified by its frequent loss in glioblastomas (6), but subsequent studies have shown that PTEN is commonly mutated in prostate cancer (7-12). In prostate tumors, the loss of PTEN occurs late in the tumorigenic pro...

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Section: Methodsmentioning

confidence: 99%

FOXO Proteins Regulate Tumor Necrosis Factor-related Apoptosis Inducing Ligand Expression

Modur

Nagarajan

Evers

et al. 2002

Journal of Biological Chemistry

340

299

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“…[1] Asymmetric cyanine dyes, such as oxazole yellow (YO and its dimer YOYO), thiazole orange (TO and its dimer TOTO), SYBR Green or PicoGreen are particularly interesting because of their extraordinary increase in fluorescence upon binding to nucleic acids. [2][3][4][5][6] This property has led to advancements in countless applications in molecular biology, such as DNA quantification in the homogenous phase and in gels, real-time polymerase chain reaction, and in DNA-binding and DNAdamage assays. Binding of cyanine dyes to nucleic acids has been widely investigated, and oxazole yellow and thiazole orange are probably amongst the most studied asymmetric cyanines.…”

Section: Introductionmentioning

confidence: 99%

“…However, the slow decay process also showed high hybridization-induced responses (decrease in decay time by 0.12-1.03 ns and increase in amplitude by factors of [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18]. Although the fast decay process showed relatively little sensitivity to double-strand formation, it was this decay along with the slow decay process that exhibited the highest responsiveness to the presence of single-mismatched stacking partners.…”

mentioning

confidence: 99%

Exploring Base‐Pair‐Specific Optical Properties of the DNA Stain Thiazole Orange

Jarikote

Krebs

Tannert

et al. 2006

Chemistry A European J

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Double‐stranded DNA offers multiple binding sites to DNA stains. Measurements of noncovalently bound dye–nucleic acid complexes are, necessarily, measurements of an ensemble of chromophores. Thus, it is difficult to assign fluorescence properties to base‐pair‐specific binding modes of cyanine dyes or, vice versa, to obtain information about the local environment of cyanines in nucleic acids by using optical spectroscopy. The feasibility to stain DNA and simultaneously probe local perturbations by optical spectroscopy would be a valuable asset to nucleic acid research. So‐called FIT probes (forced intercalation probes) were used to pinpoint the location of the DNA stain thiazole orange (TO) in PNA⋅DNA duplexes. A detailed analysis of the base‐pair dependence of optical properties is provided and enforced binding of TO is compared with “classical” binding of free TO‐PRO1. UV‐visible absorbance, circular dichroism (CD) and fluorescence spectroscopy, and melting‐curve analyses confirmed site‐specific TO intercalation. Thiazole orange exhibited base‐specific responses that are not observed in noncovalent dye–nucleic acid complexes, such as an extraordinary dependence of the TO extinction coefficient (±60 % variation of the averaged εmax of 57 000 M−1 cm−1) on nearest‐neighbor base pairs. TO signals hybridization, as shown by increases in the steady‐state fluorescence emission. Studies of TO fluorescence lifetimes in FIT–PNA and in DNA⋅DNA and PNA⋅DNA complexes highlighted four different fluorescence‐decay processes that may be closed or opened in response to matched or single‐mismatched hybridization. A very fast decay process (0.04–0.07 ns) and a slow decay process (2.33–3.95 ns) provide reliable monitors of hybridization, and the opening of a fast decay channel (0.22–0.48 ns) that resulted in an attenuation of the fluorescence emission is observed upon the formation of mismatched base pairs.

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“…For highly quantitative results the TaqMan probe method (36) is the preferred approach. Alternatively, to reduce the cost of this expensive technique, SyBr Green can also be used to label PCR products (67). The advantages of the PCR gene expression analysis lies in the speed of the technique as well as the quantitative data obtained.…”

Section: Rapid and Quantitative Methods For Validating Microarray Genmentioning

confidence: 99%

Toxicogenomics in Drug Development

et al. 2003

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Toxicogenomics represents the merging of toxicology with technologies that have been developed, together with bioinformatics, to identify and quantify global gene expression changes. It represents a new paradigm in drug development and risk assessment, which promises to generate a wealth of information towards an increased understanding of the molecular mechanisms that lead to drug toxicity and efficacy, and of DNA polymorphisms responsible for individual susceptibility to toxicity. Gene expression profiling, through the use of DNA microarray and proteomic technologies will aid in establishing links between expression profiles, mode of action and traditional toxic endpoints. Such patterns of gene expression, or 'molecular fingerprints' could be used as diagnostic or predictive markers of exposure, that is characteristic of a specific mechanism of induction of that toxic or efficacious effect. It is anticipated that toxicogenomics will be increasingly integrated into all phases of the drug development process particularly in mechanistic and predictive toxicology, and biomarker discovery. This review provides an overview of the expression profiling technologies applied in toxicogenomics, and discusses the promises as well as the future challenges of applying this discipline to the drug development process.Keywords. DNA microarrays; PCR; bioinformatics; gene expression profiling; genomics; proteomics; biomarkers; toxicology. INTRODUCTIONThe evolution of new innovative technologies in parallel with recent dramatic increases in genomic knowledge is anticipated to revolutionize toxicological studies by providing significant advances in the understanding and prediction of the toxicity and efficacy of new drugs (38, 39, 52). In classic toxicology, potential adverse effects resulting from drug exposure are evaluated using endpoints such as body and organ weight changes, biochemical and histopathological observations. Such observations, however, do not provide information about a drug's mode of action. To better evaluate the adverse effects associated with drug exposure, one needs to understand the drug's specific mode of action. Drugs are expected to induce a multitude of complex molecular perturbations in a wide variety of pathways, involving differential gene expression at the transcript and functional protein level, leading to efficacious and/or pathological outcomes. These changes in gene expression are often more sensitive and characteristic of the toxic response or process than currently employed endpoints of pathology, and have the potential to indicate toxicity already at lower doses or at earlier time points.Technological advances derived from genomic research have made it possible to follow transcriptional and translational events of genes and even the entire genome. The application of genome-wide expression profiling technologies to toxicology has created a new subdiscipline coined 'toxicogenomics,' which has the potential to provide a more comprehensive understanding of the mechanisms of pharmacology ...

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Quantitative detection of reverse transcriptase-PCR products by means of a novel and sensitive DNA stain.

Cited by 189 publications

References 10 publications

FOXO Proteins Regulate Tumor Necrosis Factor-related Apoptosis Inducing Ligand Expression

FOXO Proteins Regulate Tumor Necrosis Factor-related Apoptosis Inducing Ligand Expression

Exploring Base‐Pair‐Specific Optical Properties of the DNA Stain Thiazole Orange

Toxicogenomics in Drug Development

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