[The use of real-time RT-PCR method for the determination of Toll-like genes expression at mRNA level].

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(-deltadeltaCt) 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 ofreal-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, < 3.2% for TLR2 and < 2.84% for TLR4.

Conclusions: Based on obtained results, the optimal conditions for stimulation were: 10 microg/ml/24h for LPS, 1 microg/ml/6h for Pam3CSK4 and 1250 MeV infectious particles/24h.