2.4. Quantitative real-time PCR analysis

To detect the effects of punicalagin on gene expression in LPS-stimulated cells, bEECs were pre-incubated in six-well plates (1×10⁶ cells/well) and pretreated with punicalagin (5, 10, and 20 µg/ml) for 2 h prior to LPS (30 µg/ml) treatment in an incubator at 37 °C and 5% CO₂ for 3, 6, 9, or 12 h. Total RNA was isolated using a phenol and guanidine isothiocyanate-based TRIzol reagent according to the manufacturer’s instructions. The concentration and integrity of total RNA were measured at a 260/280 nm ratio. Quantitative polymerase chain reaction (PCR) analysis was performed using the DNA Engine Mx3000P^® (Agilent, Santa Clara, CA, USA) fluorescence detection system against a double-stranded DNA-specific fluorescent dye (Stratagene, La Jolla, CA, USA) according to optimized PCR protocols. β-Actin was amplified in parallel with the target genes and used as a normalization control. The cycling conditions were as follows: 95 °C for 10 min, followed by 40 cycles of 95 °C for 30 s, 60 °C for 60 s, and 72 °C for 60 s. Expression levels were determined using the relative threshold cycle (Ct) method as described by the manufacturer (Stratagene). The PCR reaction system (25 µl in total) contained 12.5 µl of SYBR Green PCR mix, 0.375 µl of reference dye, 1 µl of each primer (both, 10 μmol/L), 1 µl of complementary DNA (cDNA) template, and 9.125 µl of diethyl phosphorocyanidate (DEPC)-treated water. Table ​Table11 lists the gene-specific oligonucleotide primers used for real-time PCR (RT-PCR).

Primers for RT-PCR