Real-Time Quantitative PCR (RT-qPCR).
mRNA levels of IL-1β, IL-6, TNF-α, IL-10, α-SMA, Collagen I,Collagen
III, MMP-2, MMP-9, IkBα, p65 and PPAR-γ in CFs were evaluated by
RT-qPCR. Total RNA was extracted from CFs using Trizol reagent
(Invitrogen, Carlsbad, CA, USA) and the 1st strand cDNA was synthesized
using a commercial kit (Takara, Otsu, Japan) according to the producer’s
instructions. Real-time PCR (RT-PCR) was completed on a 7500 real-time
PCR system (Applied Biosystems, Carlsbad, CA, USA). The primers used in
the present work were presented in Table 1. The mRNA levels of these
targets were calculated using the 2−△△Ct method and
normalized against β-actin which was used as an internal reference gene.
The results were expressed as fold changes to control.
Table 1: Primers sequences for quantitative real-time PCR
(qRT-PCR)