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A quantitative real time PCR method to analyze T cell receptor Vb subgroup expansion by staphylococcal superantigens | Seo et al. Journal of Translational Medicine 2010 8 2 http www.translational-medicine.eom content 8 1 2 JOURNAL OF TRANSLATIONAL MEDICINE METHODOLOGY Open Access A quantitative real time PCR method to analyze T cell receptor vp subgroup expansion by staphylococcal superantigens Keun Seok Seo1 Joo Youn Park2 David S Terman3 Gregory A Bohach1 Abstract Background Staphylococcal enterotoxins SEs SE-like SEl toxins and toxic shock syndrome toxin-1 TSST-1 produced by Staphylococcus aureus belong to the subgroup of microbial superantigens SAgs . SAgs induce clonal proliferation ofT cells bearing specific variable regions of the T cell receptor b chain Vb . Quantitative real time PCR qRT-PCR has become widely accepted for rapid and reproducible mRNA quantification. Although the quantification of Vb subgroups using qRT-PCR has been reported qRT-PCR using both primers annealing to selected Vb nucleotide sequences and SYBR Green I reporter has not been applied to assess Vb-dependent expansion of T cells by SAgs. Methods Human peripheral blood mononuclear cells were stimulated with various SAgs or a monoclonal antibody specific to human CD3. Highly specific expansion of Vb subgroups was assessed by qRT-PCR using SYBR Green I reporter and primers corresponding to selected Vb nucleotide sequences. Results qRT-PCR specificities were confirmed by sequencing amplified PCR products and melting curve analysis. To assess qRT-PCR efficiencies standard curves were generated for each primer set. The average slope and R2 of standard curves were -3.3764 0.0245 and 0.99856 0.000478 respectively demonstrating that the qRT-PCR established in this study is highly efficient. With some exceptions SAg Vb specificities observed in this study were similar to those reported in previous studies. Conclusions The qRT-PCR method established in this study produced an accurate and reproducible assessment of Vb-dependent expansion of human T cells by staphylococcal SAgs. This method could be a useful tool
