FIGURE 5.

Quantification of differentially expressed defensin mRNAs by RT-PCR. A) Specific primers and annealing temperatures employed. B) RT-PCR for hBD2, hBD4, and β-actin were carried out from cell culture samples divided in five groups: basal (lane 1); cells treated with 1 μg/ml LPS for 24 h (lane 2); cells treated with TFF3 at 10 μg/ml (lane 3’) or 50 μg/ml (lane 4) for 48 h; and cells treated with vehicle (lane 5). The PCR-products were run on 2% agarose gel electrophoresis. Control reactions without reverse transcriptase were carried out. PCR was performed in a final volume of 25 μl containing 1 μl of the reverse transcription reaction, 50 μM of dNTPs, 1.5 mM MgCl2, 50 mM Tris-HCl (pH 8.0), 1 IU Taq polymerase, and 0.2 μM each of sense and antisense primers. Specific PCR for a constitutively expressed gene (β-actin) was carried out as a positive control. The relative amount of product was quantified by densitometric analysis of DNA bands (C). Defensin mRNA expression levels are shown normalized to β-actin. Results are mean ± SEM of three independent experiments.