Cdna rt premix kit

Total RNA was extracted from colonic tissue using TRIzol (Invitrogen Life Technologies, Carlsbad, CA, United States) according to the manufacturer’s instructions. The quality and quantity of crude RNA were examined using a NanoPhotometer (P330-IMPLEN, Munich, Germany). RNA was stored at −80°C for further analysis using RT-qPCR. An equal quantity of each RNA sample was reverse transcribed using an oligo deoxythymine 18-mer primer (Thermo Scientific, Waltham, MA, United States), and cDNA was synthesized using a cDNA RT PreMix kit (Bioneer, Daejeon, South Korea). Quantitative analysis of the mRNA expression of GPR41, GPR43, GPR120, IL-10, IL-13, TNF-α, IL-1β, IL-6, MCP-1 and glyceraldehyde-3-phosphatase dehydrogenase (GAPDH) was performed using RT-qPCR with a Light Cycler 480^™ platform (Roche, Roche Applied Science, Mannheim, Germany) using a SYBR Green Real-time PCR Master Mix (Toyobo, Osaka, Japan). Data processing and analysis were performed using a dedicated Light Cycler software (Roche Applied Science, version 1.2). The Ct values were normalized using GAPDH, and the relative gene expression was quantified using the standard 2^−△△Ct method. Primer sequences are listed in Supplementary Table 1.

The total RNA was extracted from the tissues using a TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, United States) according to the manufacturer’s instructions. For RT-qPCR, an equal quantity of each RNA sample (1 μg) was reverse transcribed to synthesize the cDNA using the oligo deoxythymine 18-mer ([dT]₁₈) primer (Thermo Scientific, Waltham, Massachusetts, United States) and cDNA RT PreMix kit (Bioneer, Daejeon, Republic of Korea). The qPCR analysis was performed on a Light Cycler 480™ platform (Roche Applied Science, Mannheim, Germany) in a 96-well plate using an SYBR Green Real-time PCR Master Mix (Toyobo, Osaka, Japan). Subsequently, melting curve analysis was performed to verify the purity and specificity of the amplicon. The data processing and analyses were performed using dedicated Light Cycler software supplied by the instrument manufacturer (Roche Applied Science, version 1.2). The C_t values were normalized using glyceraldehyde-3-phosphatase dehydrogenase (GAPDH) as a housekeeping gene, and the relative gene expression was quantified using the standard 2^−ΔΔCt method. The Supplementary Table 2 lists the primer sequences.