FIG. 4.

Negative regulation of TLR mRNA in human gingiva from subjects with CP. Eight of the tissues used for immunohistochemistry were randomly selected and bisected for total RNA extraction as described in Materials and Methods, along with four additional samples form healthy and diseased subjects. The total RNA was normalized among all samples, and real-time RT-PCR was used to quantitate TLR2 (A), TLR4 (B), TLR5 (C), CD14 (D), and MD2 (E) mRNA expression levels, along with IL-1β as an inflammatory marker (F). For each transcript, conventional PCR-amplified products (cleaned, with concentrations determined at OD₂₆₀) were used as standards (from 0.1 to 0.00001 ng) to generate a standard curve for absolute real-time quantitation. The absolute mRNA levels of all the genes were then normalized to β-actin levels of individual tissue samples. All quantitations were performed in triplicate. The normalized initial concentration of each transcript in every sample was converted to the initial copy number (see Materials and Methods). Error bars indicate standard error. The level of mRNA for TLR2 in diseased samples was statistically different from that in samples from healthy controls (P < 0.05, Student's t test).