Tissue Expression of Human Toll-Like Receptors and Differential Regulation of Toll-Like Receptor mRNAs in Leukocytes in Response to Microbes, Their Products, and Cytokines

The software package Primer Express (PE Applied Biosystems) was used to identify appropriate TaqMan primer sets. Forward and reverse probes were chosen to have a melting temperature of about 60°C and the probes to have a melting temperature of about 70°C. Probes were selected that had a greater number of cytosine than guanine nucleotides. Amplification primers were further restricted to those containing at least 60% A/T in the 3′ ends. All probes were labeled on the 5′ end with FAM as a reporter and on the 3′ end with TAMRA as the quenching dye with the exception of the GAPDH probe which was labeled on the 5′ end with 2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein (JOE). All sequences are presented in the 5′→3′ direction.