title: [The use of real-time RT-PCR method for the determination of Toll-like genes expression at mRNA level].
abstract: BACKGROUND
  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.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Cześcik
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Trzcińska
authors.forename: Milena
authors.initials: M
authors.lastname: Dunal-Szczepaniak
authors.forename: Joanna
authors.initials: J
authors.lastname: Siennicka
mesh_terms: D015870:Gene Expression; D006801:Humans; D007113:Immunity, Innate / Q000235:genetics*; D007963:Leukocytes, Mononuclear / Q000276:immunology*; D012333:RNA, Messenger / Q000032:analysis*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D051193:Toll-Like Receptors / Q000235:genetics*
chemical_list: D012333:RNA, Messenger; D051193:Toll-Like Receptors