Quantifying Gene Expression via RT-PCR

Total RNA was isolated using a Sangon UNIQ-10 column Trizol total RNA extraction kit according to the instructions of the manufacturer. RNA was reversely transcribed using an ImProm-II Reverse Transcription System cDNA synthesis kit (Promega Corporation, Madison, WI, USA). The real-time reverse transcription polymerase chain reaction (RT-PCR) oligonucleotide primers used for rat iNOS, TNF-α, and IL-1β are shown in Table 1. The reactions were set up in duplicates in 25 μL total volumes with 1 μL of each primer (0.3 μM final concentrations), 12.5 μL of FastStart Universal SYBR Green Master (ROX) (Roche), and 1 μL of template. The PCR cycle was as follows: 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds, 60°C for 1 minute, and a melt curve analysis was performed at the end of each experiment to verify that a single product per primer pair was amplified. The amplification and analysis were performed using an ABI Prism 7,500 real-time PCR system. Samples were analyzed using the relative C_T method. The fold increase or decrease was determined relative to a blank control after normalizing to a housekeeping gene using 2^−ΔΔC_T.28 (link),29 (link)