Reverse Transcription and Real Time PCR (RT2PCR)-RNA was prepared from cells left untreated, stimulated with TNFα (10 ng/ml) or flagellin (1 μg/ml) for 3 h. RT2PCR was performed with an iCycler (Bio-Rad) to quantify SYBR-green labeled products generated from PCR products of 1st strand cDNA prepared from TLR1 through TLR10 mRNA, 18S rRNA, and GAPDH mRNA. RT2PCR (25 ul reaction volume) was performed with the appropriate primers (SuperArray) in triplicate with HotStart Taq DNA polymerase (SuperArray) at 95°C for 5 min to activate Taq and amplified for 40 cycles (95°C, 30 sec, 55°C, 30 sec, 72°C, 30 sec). RT2PCR was performed on the minus RT controls with TLR5 primers to detect DNA contamination. Fold change in mRNA expression was expressed as 2ΔΔCt. ΔCt is the difference in threshold cycles for the TLR mRNAs and 18S rRNA. ΔΔCt is the difference between ΔCt non-simulated control and ΔCt stimulated sample.