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Medycyna Doświadczalna i Mikrobiologia 2014, 66(1): 17-22

Zastosowanie metody real-time RT-PCR do oznaczania ekspresji genów receptorów Toll-like na poziomie mRNA
[The use of real-time RT-PCR method for the determination of Toll-like genes expression at mRNA level]

A. Częścik, A. Trzcińska, M. Dunal-Szczepaniak, J. Siennicka

Streszczenie

W pracy przedstawiono metodykę doświadczenia mającego na celu optymalizację warunków stymulacji komórek PBMC i walidację metody real-time RT-PCR stosowanej do oznaczania ekspresji na poziomie mRNA receptorów Toll-like w tych komórkach.

Abstract

Introduction: Toll-like receptors (TLRs) are an important component of a innate immune system. Stimulation of TLRs, through action with helper T cells, could change Th1/Th2 balance and thus affect adaptive immune response. The aim of this work was to optimize the
stimulation of the peripheral blood mononuclear cells (PBMC) and the validation of real-time RT-PCR method for determination of Toll-like gene expression in these cells. Methods: PBMCs from healthy donors were stimulated with measles viruses and ligands for
TLR2 and TLR4. For examinations the real time RT-PCR (QuantiFast®Assay, Qiagen) was used. Fold change of TLRs expression was normalized to GAPDH and estimated by 2-ΔΔCt method. Validation of real-time RT-PCR method was performed for repeatability and efficiency. Results: The level of gene expression varies between individuals and was dose and time of incubation dependent. The efficiency of real-time RT-PCR was 90.4% ± 10.2 for GAPDH, 87.0% ± 8.2 for TLR2 and 44.5% ± 9.2 for TLR4. Repeatability, expressed as relative standard deviation (RSD) for Ct values was less than 0.70% for GAPDH, and

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