Quantification of cytokine messenger RNA in transfected human T cells by RT-PCR and an automated electrochemiluminescence-based post-PCR detection system

Motmans, Kris; Raus, Jef; Vandevyver, Caroline

doi:10.1016/0022-1759(95)00259-6

Cited by 34 publications

(8 citation statements)

References 13 publications

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“…These studies are focused on detecting, discovering the causes for, and finding cures for diseases. [339][340][341][342][343][344][345][346][347][348][349][350][351][352] A rapidly expanding area of clinical diagnostics is the use of molecular diagnostics or molecular probe assays (called molecular assays for simplicity). Typically, these involve the hybridization and labeling of specific nucleic acid sequences to test for disease states, predisposition for a disease and infectious diseases.…”

Section: Clinical Applicationsmentioning

confidence: 99%

ECL—Electrochemical luminescence

Pyati

Richter

2007

Annu. Rep. Prog. Chem., Sect. C: Phys. Chem.

121

146

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Section: Clinical Applicationsmentioning

confidence: 99%

ECL—Electrochemical luminescence

Pyati

Richter

2007

Annu. Rep. Prog. Chem., Sect. C: Phys. Chem.

121

146

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“…The incorporation procedure is a relatively reliable method, its success rate is over 80%. 21 We used different concentrations of standard TBR solutions to get the evolution of the ECL intensities and the TBR concentrations. Then we detected the ECL intensities from the modified electrode and calculated the amount of TBR modified on the electrode.…”

Section: Ministry Of Education Key Laboratory Of Analysis and Detectionmentioning

confidence: 99%

A sensitive and specific electrochemiluminescent sensor for lead based on DNAzyme

et al. 2009

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“…Non-competitive RT-PCR relies exclusively on the linear relationship between the starting amount of target RNA and the final quantity of product formed during PCR before reaching a plateau phase of the amplification. Different approaches have been described to obtain quantification of products generated by non-competitive RT-PCR, such as radio-or chemiluminescence labelling of either the PCR product itself or a probe hybridized to it, respectively (11)(12)(13). These attempts, however, are cost and labour intense.…”

Section: Quantitative Assessment Of the Expression Of Melanoma-associmentioning

confidence: 99%

Quantitative assessment of the expression of melanoma-associated antigens by non-competitive reverse transcription polymerase chain reaction

et al. 2001

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The assessment of tumor-associated antigens (TAA) recognized by T lymphocytes is a prerequisite for diagnosis and immunotherapy of melanoma. Different reverse transcription-polymerase chain reaction (RT-PCR) protocols allowing the quantification of the TAA mRNA expression in the solid tumor or the detection of circulating melanoma cells have been described. We have recently shown a positive correlation between the amount of specific product formed by RT-PCR and the staining intensity in immunohistochemical analysis of the corresponding sample. Here we describe a quantification procedure based on the direct digitization of the PCR products after separation on ethidium bromide-stained agarose gels, followed by computer-assisted densitometry. To standardize our method, we examined the linear range of the densitometric quantification procedure as reflected by the correlation of signal intensity to the amount of the corresponding DNA. As an internal measure for the so-termed 'effective' cDNA in the different samples after RNA isolation and reverse transcription, a ß-actin PCR was introduced. Subsequently, we chose four sets of primers for the melanomaassociated antigens MAGE1, tyrosinase, Melan A/MART-1 and gp100/Pmel17 and performed PCR analysis over a range of cycle numbers. In each case, the amplification rate remained constant up to at least 26 cycles under the respective conditions. Plotting the logarithm of the amount of product against the cycle number yields a slope that equals the logarithm of the amplification rate. The amount of starting material can be determined from the intercept with the ordinate. In summary, the method introduced in the present work allows the quantification of TAA in melanoma which might be important for the monitoring of disease. Technically the method is sound and sensitive, avoids post-PCR manipulations and can be performed with the standard equipment of a molecular biology laboratory. It can be applied also to other solid tumors and leukemias.

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Quantification of cytokine messenger RNA in transfected human T cells by RT-PCR and an automated electrochemiluminescence-based post-PCR detection system

Cited by 34 publications

References 13 publications

ECL—Electrochemical luminescence

ECL—Electrochemical luminescence

A sensitive and specific electrochemiluminescent sensor for lead based on DNAzyme

Quantitative assessment of the expression of melanoma-associated antigens by non-competitive reverse transcription polymerase chain reaction

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