QUANTIFICATION OF MURINE CYTOKINE mRNAs USING REAL TIME QUANTITATIVE REVERSE TRANSCRIPTASE PCR

Overbergh, Lutgart; Valckx, Dirk; Waer, Mark; Mathieu, Chantal

doi:10.1006/cyto.1998.0426

Cited by 527 publications

(362 citation statements)

References 22 publications

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“…Each run included a negative control and a known GAPDH control. Results from real-time PCR were calculated as threshold cycles normalized to that of the GAPDH gene according to the method of Overbergh et al, (1999) and expressed as DC t values. Expression of HSulf-1 by semi-quantitative RT-PCR was as previously described (Lai et al, 2003).…”

Section: Cell Culturementioning

confidence: 99%

Epigenetic silencing of HSulf-1 in ovarian cancer:implications in chemoresistance

et al. 2007

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To investigate the mechanism by which HSulf-1 expression is downregulated in ovarian cancer, DNA methylation and histone acetylation of HSulf-1 was analysed in ovarian cancer cell lines and primary tumors. Treatment of OV207 and SKOV3 by 5-aza-2 0 -deoxycytidine resulted in increased transcription of HSulf-1. Sequence analysis of bisulfite-modified genomic DNA from ovarian cell lines and primary tumors without HSulf-1 expression revealed an increase in the frequency of methylation of 12 CpG sites in exon 1A. Chromatin immunoprecipitation assays showed an increase in histone H3 methylation in cell lines without HSulf-1 expression. To assess the significance of HSulf-1 downregulation in ovarian cancer, OV167 and OV202 cells were transfected with HSulf-1 siRNA. Downregulation of HSulf-1 expression in OV167 and OV202 cells lead to an attenuation of cisplatin-induced cytotoxicity. Moreover, patients with ovarian tumors expressing higher levels of HSulf-1 showed a 90% response rate (27/30) to chemotherapy compared to a response rate of 63% (19/30) in those with weak or moderate levels (P ¼ 0.0146, v 2 test). Collectively, these data indicate that HSulf-1 is epigenetically silenced in ovarian cancer and that epigenetic therapy targeting HSulf-1 might sensitize ovarian tumors to conventional first-line therapies.

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Section: Cell Culturementioning

confidence: 99%

Epigenetic silencing of HSulf-1 in ovarian cancer:implications in chemoresistance

et al. 2007

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“…Total RNA was isolated from fresh pancreatic islets (n=200) using the High Pure RNA isolation kit (Roche Diagnostics, Indianapolis, Ind., USA) resulting in DNA-free RNA, while for total RNA extraction from the islet grafts and the control kidney tissue TRIzol Liquid Suspension reagent (Life Technologies, Gaithersburg, Md., USA) was used. After reverse transcription with Superscript II reverse transcriptase (Life Technologies), real-time quantitative PCR was done in the ABI Prism 7700 Sequence Detector (Perkin Elmer/Applied Biosystems, Foster City, Calif., USA) for IL-1β, IFN-γ, IL-15, fractalkine, IP-10 and MIP-3α [41]. PCR amplification reactions (25 µl) contained 0.5 µl cDNA sample, 2.5 µl 10× TaqMan Buffer A, 200 µmol/l dNTPs, 5 mmol/l MgCl 2 , 0.625 U AmpliTaq Gold, 200 to 300 nmol/l of primerforward (F) and 200 to 900 nmol/l of primer-reverse (R) ( Table 1).…”

Section: Isolation and Transplantation Of Islets From Nod Micementioning

confidence: 99%

“…A normalisation to β-actin (in vivo experiments) or GAPDH (in vitro experiments) as "housekeeping" genes was done for each sample. The method used for quantification by real time PCR is the standard-curve method, as described in previous publications by our group [41,42].…”

Section: Isolation and Transplantation Of Islets From Nod Micementioning

confidence: 99%

IL-1β and IFN-γ induce the expression of diverse chemokines and IL-15 in human and rat pancreatic islet cells, and in islets from pre-diabetic NOD mice

et al. 2003

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Aims/hypothesis. Cytokines and chemokines are important mediators of immune responses due to their ability to recruit and activate leukocytes. Using microarray analysis we observed that rat beta cells exposed to IL-1β and IFN-γ have increased mRNA levels of chemokines and IL-15. The aim of this study was to characterize the expression of IP-10, MIP-3α, fractalkine and IL-15 in rat beta cells, human pancreatic islets, and in islets isolated from NOD mice, both during the pre-diabetic period and following islet transplantation. Methods. FACS-purified rat beta cells and human islets were cultured with IL-1β, IFN-γ and/or TNF-α. Islets were isolated from NOD or BALB/c mice at different ages. For syngeneic islet transplantation, 2-or 3-week-old NOD islets were grafted under the kidney capsule of spontaneously diabetic NOD recipients. Chemokine and IL-15 mRNA expression and protein release were evaluated, respectively, by RT-PCR and ELISA.Results. Human islets and rat beta cells express IP-10, MIP-3α, fractalkine and IL-15 mRNAs upon exposure to cytokines. The expression of IL-15, IP-10 and fractalkine is regulated by IFN-γ, while the expression of MIP-3α is IL-1β-dependent. Moreover, cytokines induced IL-15, IP-10, Mig, I-TAC and MIP-3α protein accumulation in culture medium from human islets. In vivo, there was an age-related increase in IL-15, IP-10 and MIP-3α expression in islets isolated from NOD mice. Following syngeneic islet transplantation, increased expression of IL-1β, IFN-γ, fractalkine, IP-10, MCP-1 and MIP-3α mRNAs were observed in the grafts. Conclusion/interpretation. Cytokine-exposed islets or beta cells express chemokines and IL-15. This could contribute to the recruitment and activation of mononuclear cells and development of insulitis in early Type 1 diabetes and during graft destruction. [Diabetologia (2003) 46:255-266] Keywords Type 1 diabetes mellitus, chemokines, IL-15, NOD mice, beta cells, interferon-γ, interleukin-1β, pancreatic islets, islet transplantation, insulitis.

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“…For murine ␤-actin detection, ␤-actin primers and probe were used, as previously described. 49 To correct for differences in DNA recovery from samples, the data are presented as the ratio of Ad genome copies to ␤-actin DNA copies.…”

Section: Gene Therapymentioning

confidence: 99%

Influence of adenoviral fiber mutations on viral encapsidation, infectivity and in vivo tropism

et al. 2001

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Targeting of adenovirus (Ad)-encoded therapeutic genes to specific cell types has become a major goal in gene therapy. Redirecting the specificity of infection requires the abrogation of the natural interaction between the viral fiber and its cellular receptors (CAR) and the simultaneous introduction of a new binding specificity into the viral capsid. To abrogate the natural affinity of the fiber, we have mutated residues presumed to be directly or indirectly involved in CARbinding in the knob domain of the fiber protein. These residues are located in the AB loop (Ser408) and in the DG loop (Tyr491, Ala494, Ala503). The mutations Ser408Glu,

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QUANTIFICATION OF MURINE CYTOKINE mRNAs USING REAL TIME QUANTITATIVE REVERSE TRANSCRIPTASE PCR

Cited by 527 publications

References 22 publications

Epigenetic silencing of HSulf-1 in ovarian cancer:implications in chemoresistance

Epigenetic silencing of HSulf-1 in ovarian cancer:implications in chemoresistance

IL-1β and IFN-γ induce the expression of diverse chemokines and IL-15 in human and rat pancreatic islet cells, and in islets from pre-diabetic NOD mice

Influence of adenoviral fiber mutations on viral encapsidation, infectivity and in vivo tropism

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