scholarly journals Establishment and Validation of a Non-Radioactive Method for In Vitro Transcription Assay Using Primer Extension and Quantitative Real Time PCR

PLoS ONE ◽  
2015 ◽  
Vol 10 (8) ◽  
pp. e0135317 ◽  
Author(s):  
Juan Wang ◽  
Shasha Zhao ◽  
Ying Zhou ◽  
Yun Wei ◽  
Wensheng Deng
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
In Vitro Transcription ◽  
Primer Extension ◽  
Quantitative Real Time Pcr ◽  
Transcription Assay

scholarly journals The circRAB3IP Mediated by eIF4A3 and LEF1 Contributes to Enzalutamide Resistance in Prostate Cancer by Targeting miR-133a-3p/miR-133b/SGK1 Pathway

2021 ◽  
Vol 11 ◽  
Author(s):  
Dong Chen ◽  
Yaqin Wang ◽  
Feiya Yang ◽  
Adili Keranmu ◽  
Qingxin Zhao ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Real Time ◽  
Expression Pattern ◽  
Real Time Pcr ◽  
Bioinformatic Analysis ◽  
Biological Functions ◽  
Quantitative Real Time Pcr ◽  
Regulatory Effects

An increasing number of studies have shown that circRNAs are closely related to the carcinogenesis and development of prostate cancer (PCa). However, little is known about the effect of the biological functions of circRNAs on the enzalutamide resistance of PCa. Through bioinformatic analysis and experiments, we investigated the expression pattern of circRNAs in enzalutamide-resistant PCa cells. Quantitative real-time PCR was used to detect the expression of circRAB3IP, and plasmids that knock down or overexpress circRAB3IP were used to evaluate its effect on the enzalutamide sensitivity of PCa cells. Mechanistically, we explored the potential regulatory effects of eIF4A3 and LEF1 on the biogenesis of circRAB3IP. Our in vivo and in vitro data indicated that increased expression of circRAB3IP was found in enzalutamide-resistant PCa, and knockdown of circRAB3IP significantly enhanced enzalutamide sensitivity in PCa cells. However, upregulation of circRAB3IP resulted in the opposite effects. Further mechanistic research demonstrated that circRAB3IP could regulate the expression of serum and glucocorticoid-regulated kinase 1 (SGK1) by serving as a sponge that directly targets miR-133a-3p/miR-133b. Then, we showed that circRAB3IP partially exerted its biological functions via SGK1 signaling. Furthermore, we discovered that eIF4A3 and LEF1 might increase circRAB3IP expression in PCa.

scholarly journals Use of quantitative real-time PCR for studying the dissemination of Leptospira interrogans in the guinea pig infection model of leptospirosis

2009 ◽  
Vol 58 (5) ◽  
pp. 648-655 ◽  
Author(s):  
Kristel Lourdault ◽  
Florence Aviat ◽  
Mathieu Picardeau
Keyword(s):  
Guinea Pig ◽  
Real Time ◽  
Real Time Pcr ◽  
Guinea Pigs ◽  
Infection Model ◽  
Avirulent Strain ◽  
Leptospira Interrogans ◽  
Quantitative Real Time Pcr ◽  
Guinea Pig Model

The dynamics of leptospirosis infection have been poorly studied. The purpose of this study was to determine the LD50, rate of bacterial dissemination, histopathology and antibody responses against leptospira following inoculation with the highly virulent Leptospira interrogans Fiocruz L1-130 strain in a guinea pig model of leptospirosis. Three routes of infection (intraperitoneal, conjunctival and subcutaneous inoculation) were used to establish disease in guinea pigs. The size and kinetics of leptospiral burdens in the blood and tissues of infected animals were determined over a 1 week course of infection using quantitative real-time PCR (qPCR). Bacteraemia peaked at day 5 post-infection reaching more than 5×104 leptospires ml−1. The highest spirochaetal load was found in the liver and kidneys, and was associated with alterations in organ tissues and a decline in liver and kidney functions. In contrast, lesions and bacteria were not detected in guinea pigs infected with an avirulent strain derived from a high-passage-number in vitro-passaged variant of the Fiocruz L1-130 strain. The use of qPCR supports the findings of earlier studies and provides an easy and reliable method for the quantification of L. interrogans in the tissues of infected animals. qPCR will be used in future studies to evaluate the efficacy of vaccine candidates against leptospirosis and the virulence of selected L. interrogans mutants relative to the parental strain.

scholarly journals Validation of housekeeping genes for quantitative real-time PCR in in-vivo and in-vitro models of cerebral ischaemia

2009 ◽  
Vol 10 (1) ◽  
pp. 57 ◽  
Author(s):  
Carme Gubern ◽  
Olivia Hurtado ◽  
Rocío Rodríguez ◽  
Jesús R Morales ◽  
Víctor G Romera ◽  
...  
Keyword(s):  
Real Time ◽  
Cerebral Ischaemia ◽  
Real Time Pcr ◽  
Housekeeping Genes ◽  
In Vitro Models ◽  
Quantitative Real Time Pcr

p29ING4 Regulates the Production of Pro-Angiogenic Molecules by Human Myeloma Cells in Normoxic and Hypoxic Conditions Being Involved in Myeloma-Induced Angiogenesis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 515-515
Author(s):  
Sara Tagliaferri ◽  
Francesca Morandi ◽  
Paolo Lunghi ◽  
Simona Colla ◽  
Mirca Lazzaretti ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Mrna Expression ◽  
Real Time ◽  
Real Time Pcr ◽  
Interleukin 8 ◽  
Mrna Levels ◽  
Quantitative Real Time Pcr ◽  
Angiogenic Switch ◽  
Hypoxic Conditions

Abstract Multiple myeloma (MM) cells produce several angiogenic molecules as VEGF, Angiopoietin-1 (Ang-1), interleukin-8 (IL-8) and osteopontin (OPN), however the molecular mechanisms underlying the angiogenic switch are not completely elucidated. The candidate tumor suppressor gene inhibitor of growth family member 4 (p29ING4) has been recently implicated in solid tumors as a repressor of angiogenesis and tumor growth through the suppression of angiogenic related molecules including interleukin-8 (IL-8) and the hypoxia inducible factor (HIF)-1 alpha. In this study we investigate the potential involvement of p29ING4 in the angiogenic switch in MM. First using quantitative real time PCR we compared p29ING4 with VEGF, Ang-1, IL-8 and OPN mRNA levels in eight human myeloma cell lines (HMCLs). A significantly negative correlation was observed between ING4 and IL-8 and a trend of correlation with OPN. Following we transfected HMCLs JJN3, OPM-2 and RPMI-8226 with specific siRNA to completely block the expression of p29ING4 checking the effect on the expression and production of the myeloma-related angiogenic molecules VEGF, Ang-1, IL-8 and OPN by quantitative real time PCR and ELISA assay. p29ING4 suppression in HMCLs did not affect VEGF and Ang-1 production but induced a strong up-regulation of IL-8 mRNA and IL-8 protein secretion. Similarly an induction of OPN mRNA expression as well as OPN secretion was induced by siRNA anti-p29ING4. Moreover conditioned media of HMCLs transfected with siRNA anti p29ING4 significantly increased vessel formation in an experimental in vitro model of angiogenesis (ANGIO kit) as compared to controls. Further we investigate the role of p29ING4 in the production of angiogenic molecule by MM cells in hypoxic condition compared to normoxic one as well as its potential relationship with HIF-1alpha system. Hypoxia induced HIF-1alpha expression at nuclear level and its activity in HMCLs and p29ING4 suppression by siRNA further induced HIF-1alpha transcriptional activity with an increase of its target gene Nip-3 in HMCLs. In turn the block of HIF1-alpha by specific siRNA up-regulated p29ING4 and suppressed IL-8 and OPN mRNA expression by HMCLs suggesting a relationship between p29ING4 and HIF-1alpha activity. These in vitro observations have been extended in vivo by the finding of a significant correlation between bone marrow (BM) plasma IL-8 levels and p29ING4 mRNA expression in purified MM cells obtained from 40 newly diagnosed MM patients (R=−0.58 Spearman’s 2-tailed test: p=0.04), consistently MM patients with higher BM IL-8 levels have a significantly lower p29ING4 mRNA levels. Similarly MM patients positive for OPN expression with high OPN BM levels had a significant lower p29ING4 levels (p=0.02). Finally we found that MM patients with high microvalscular density (MVD>30) have significant lower p29ING4 levels as compared to those with low MVD (<30) (p=0.04) and that MM patients with histological high grade had significant lower p29ING4 mRNA levels (Mann-Whitney 2-tailed: p=0.05). In conclusion, our data indicate that the tumor suppressor p29ING4 regulate the production of angiogenic molecules by MM cells both in normoxic and hypoxic conditions being involved in MM-induced angiogenesis and potentially in tumor progression.

Gene Expression Profiling in Multiple Myeloma Cells Treated with the Novel Anti-Myeloma Agents Zoledronate and Fluvastatin.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5078-5078
Author(s):  
Timothy J. Molloy ◽  
Baulch-Brown Cindy ◽  
Yi-Mo Deng ◽  
Andrew Spencer ◽  
David F. Ma
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Real Time ◽  
Real Time Pcr ◽  
Mevalonate Pathway ◽  
Annexin V ◽  
Quantitative Real Time Pcr ◽  
Myeloma Cells ◽  
Qrt Pcr

Abstract We have shown in vitro that multiple myeloma (MM) cells can be destroyed by treating them with the mevalonate pathway inhibitors zoledronate and fluvastatin. While the efficacy of these compounds singly and combination have been demonstrated, their exact modes of action remain largely unknown. The present study aimed to use microarray and quantitative real-time PCR (QRT-PCR) techniques to analyse gene expression in treated myeloma cells to identify novel genes and pathways involved in the anti-myeloma action of these compounds. The human MM cell line NCI-H929 was treated with zoledronate and fluvastatin singly and in combination, and RNA was extracted and used to interrogate oligonucleotide microarrays consisting of 19,000 features representing known and unknown genes. Quantitative real-time PCR was subsequently used to confirm the expression of several genes of interest. Flow cytometry with Annexin V FITC staining was used to detect apoptosis. It was observed that genes related to apoptosis (caspases and p53-related genes), cell cycle control (cyclins), GTPase signalling (Rabs), and growth and proliferation (growth factors) were particularly affected by zoledronate and fluvastatin, and some of these genetic effects were synergistic when a combination of zoledronate and fluvastatin was used. QRT-PCR confirmed the effects on the caspase- and p53-related apoptotic pathways, and these effects were correlated with increased apoptosis in the myeloma cells. The mevalonate pathway inhibitors fluvastatin and zoledronate are highly efficient at killing MM cells, and their effects appear to be synergistic. Our microarray and QT-PCR analyses demonstrated that the expression of specific groups of genes important to the survival and proliferation of myeloma cells are affected by these compounds. p53 and caspase-dependent pathways appear to be the key apoptotic cascades stimulated. Insights into the mechanisms of these novel therapeutics are important as they might help to define their roles in the treatment of multiple myeloma.

scholarly journals Selection of reference genes for quantitative real-time PCR in equine in vivo and fresh and frozen-thawed in vitro blastocysts

2009 ◽  
Vol 2 (1) ◽  
pp. 246 ◽  
Author(s):  
Katrien Smits ◽  
Karen Goossens ◽  
Ann Van Soom ◽  
Jan Govaere ◽  
Maarten Hoogewijs ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Reference Genes ◽  
Quantitative Real Time Pcr ◽  
Selection Of

scholarly journals Biological evaluation of transdichloridoplatinum( II) complexes with 3- and 4-acetylpyridine in comparison to cisplatin

2013 ◽  
Vol 47 (4) ◽  
pp. 346-357 ◽  
Author(s):  
Lana Filipovic ◽  
Sandra Arandelovic ◽  
Nevenka Gligorijevic ◽  
Ana Krivokuca ◽  
Radmila Jankovic ◽  
...  
Keyword(s):  
Western Blot ◽  
Mrna Expression ◽  
Real Time ◽  
Real Time Pcr ◽  
Inductively Coupled Plasma ◽  
Gelatin Zymography ◽  
Biological Evaluation ◽  
Emission Spectrometry ◽  
Quantitative Real Time Pcr

Abstract Background. In our previous study we reported the synthesis and cytotoxicity of two trans-platinum(II) complexes: trans-[PtCl2(3-acetylpyridine)2] (1) and trans-[PtCl2(4-acetylpyridine)2] (2), revealing significant cytotoxic potential of 2. In order to evaluate the mechanism underlying biological activity of both trans-Pt(II) isomers, comparative studies versus cisplatin were performed in HeLa, MRC-5 and MS1 cells. Materials and methods. The cytotoxic activity of the investigated complexes was determined using SRB assay. The colagenolytic activity was determined using gelatin zymography, while the effect of platinum complexes on matrix metalloproteinases 2 and 9 mRNA expression was evaluated by quantitative real-time PCR. Apoptotic potential and cell cycle alterations were determined by FACS analyses. Western blot analysis was used to evaluate the effect on expression of DNA-repair enzyme ERCC1, and quantitative real-time PCR was used for the ERCC1 mRNA expression analysis. In vitro antiangiogenic potential was determined by tube formation assay. Platinum content in intracellular DNA and proteins was determined by inductively coupled plasma-optical emission spectrometry. Results. Compound 2 displayed an apparent cytoselective profile, and flow cytometry analysis in HeLa cells indicated that 2 exerted antiproliferative effect through apoptosis induction, while 1 induced both apoptosis and necrosis. Action of 1 and 2, as analyzed by quantitative real-time PCR and Western blot, was associated with down-regulation of ERCC1. Both trans-complexes inhibited MMP-9 mRNA expression in HeLa, while 2 significantly abrogated in vitro tubulogenesis in MS1 cells. Conclusions. The ability of 2 to induce multiple and selective in vitro cytotoxic effects encourages further investigations of trans-platinum(II) complexes with substituted pyridines.

scholarly journals Quantification of In Vitro and In Vivo Cryptosporidium parvum Infection by Using Real-Time PCR

2006 ◽  
Vol 72 (6) ◽  
pp. 4484-4488 ◽  
Author(s):  
Nihal T. Godiwala ◽  
Alain Vandewalle ◽  
Honorine D. Ward ◽  
Brett A. Leav
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Absolute Quantification ◽  
Host Tissue ◽  
Quantitative Real Time Pcr ◽  
A New Technique ◽  
First Time ◽  
Laboratory Models

ABSTRACT Established methods for quantifying experimental Cryptosporidium infection are highly variable and subjective. We describe a new technique using quantitative real-time PCR (qPCR) that can be used to measure in vitro and in vivo laboratory infections with Cryptosporidium. We show for the first time that qPCR permits absolute quantification of the parasite while simultaneously controlling for the amount of host tissue and correlates significantly with established methods of quantification in in vitro and in vivo laboratory models of infection.

Sign in / Sign up

Export Citation Format

Share Document