Quantitative Real-Time PCR Analysis of HSV-1 and TGF-β1 Effects

Total RNA was extracted from TM cells from mock-infected or infected with HSV-1 at a MOI 1 of 1 and/or stimulated with recombinant active TGF-β1 at 15 ng/ml (0.6 nM) for 12 h using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA). The RNA was reverse transcribed using a cDNA synthesis kit (PrimeScript RT Reagent Kit, Takara Bio, Kusatsu, Japan) according to the manufacturer’s instructions. Using a Roche Diagnostics LightCycler 2.0 Real-Time PCR System (Roche, Mannheim, Germany), the relative expression levels of mRNA were determined as previously described.[10 (link)] Reactions for each sample were run in triplicate, cycle thresholds were normalized to β-actin expression, and comparative quantitation was performed (LightCycler software, version 4.1, Roche). Only individual PCR samples with single peak dissociation curves were selected for data analysis. Table 1 shows the sequences of the primer sets. To ensure equal loading and amplification, all products were normalized to a β-actin as an internal control. The results are the averages of three independent experiments.