Flagellin acting via TLR5 is the major activator of key signaling pathways leading to NF-κB and proinflammatory gene program activation in intestinal epithelial cells

Table 2 Change in TLR mRNA levels following TNFα or FliC stimulation.

ND None detected by RT²PCR
M None detected above level of minus RT control.
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 1^st 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 2^ΔΔCt. 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.

ISSN: 1471-2180