Quantitative PCR Analysis of Gene Expression

The protocol was followed by the established method [31 (link)]. RAW264.7 cells were cultured with PIMP in the presence or absence of LPS (1 μg/mL). Using the TRIzol reagent, total RNA was extracted from the colon tissue and RAW264.7 cells in accordance with the guidelines provided in the manual. Using a Transcription cDNA Synthesis Kit, the extracted RNA was reverse-transcribed into complementary DNA (cDNA). Table S1 contains primer information. The CFX96^TM system (Bio-Rad, Hercules, CA, USA) in conjunction with the FastStart SYBR Green Master Mix (Takara SYBR^® Pre-mix Ex Taq^TM II, Dalian, China) was used to conduct the PCR reactions. A typical two-step protocol was used for the PCR amplification: 40 cycles of 95 °C for 3 s and 60 °C for 30 s were followed by one cycle at 95 °C for 30 s. The samples were subjected to melting curve analysis. The internal reference for relative expression was GAPDH mRNA.