Easy blue kit

Easy Blue^® kits (Intron Biotechnology, Seoul, Korea) were used to extract cellular RNA. TOPscript™ RT Dry MIX was used to reverse-transcribe an equivalent amount of RNA (500 ng per sample), and a Thermal Cycler Dice Real-Time System (TaKaRa Bio Inc., Shiga, Japan) was used to perform quantitative real-time PCR amplification. The oligonucleotide primers which are listed in Supplementary Table S3 are designed by Primer3 and the specificity checking module uses BLAST. Amplification was measured by incorporation of TB Green^TM Premix Ex Taq (TaKaRa Bio Inc., Shiga, Japan) to detect TNF-α, IL-1β, IL-6, and β-actin mRNA expression. PCRs were carried out for 50 cycles applying the following conditions: denaturation at 95 °C for 5 s, annealing at 55 °C for 10 s, and elongation at 72 °C for 20 s. The mean Ct value of the gene of interest was calculated using triplicate measurements and normalized against the mean Ct value of the control gene, β-actin.

RNA was extracted from the distal colon tissue using Easy Blue^® kits (Intron Biotechnology, Seoul, Korea). From each sample, 500 ng of RNA was reverse-transcribed by TOPscript^™ RT Dry MIX, and qRT-PCR amplification was carried out by the incorporation of TB GreenTM Premix Ex Taq (TaKaRa) in accordance with the manufacturer’s recommended protocols. The primer sequences are listed in Table 2. A dissociation curve analysis of genes showed a single peak for each. The mean Ct of the target gene was calculated from triplicate measurements and was normalized with the mean Ct of β-actin.

The cellular RNA was extracted following the manufacturer’s instructions using the Easy Blue^® kits (Intron Biotechnology, Seoul, Republic of Korea). Total RNA was reverse transcribed using first-strand cDNA synthesis kit (Amersham Pharmacia Biotech, Oakville, ON, Canada). Real-time PCR was performed using Thermal Cycler Dice Real Time PCR System (Takara, Tokyo, Japan). The following primers were utilized for SYBR green real-time RT-PCR: for FMN1 sense primer, 5′-AGC TTT GCT CAA ATT GAC GC-3′ and antisense primer 5′-TCC CTT CGG TTC AGT TTT GG-3′; for ITGA2, sense primer, 5′-AGG AAC ATG GGA ACT GGA GG-3 and anti-sense primer, 5′-GGC TGA GTT GCA GGT GAG AA-3′; for COL13A1, sense primer, 5′-CGG TAA ACC AGG AGA CAT GG-3′ and anti-sense primer, 5′-GAA CCT CTG CTC CTG GAT TG-3′; for VEGFC, sense primer, 5′-CAG CTA AGG AAA GGA GGC TG-3′ and anti-sense primer, 5′-CCA AAC TCC TTC CCC ACA TC-3′; for NRG1, sense primer, 5′-GGA ATG GCC GAT GTG TAT GT-3′ and anti-sense primer, 5′-GAA ATG GGT GAG AGA GCC TG-3; for β-actin, sense primer, 5′-CAA ACA TGA TCT GGG TCA TC-3′ and anti-sense primer, 5′-GCT CGT CGT CGA CAA CGG CT-3′. A dissociation curve analysis showed a single peak. From three independent observations, the mean cycle threshold (Ct) of the target gene was determined and normalized using the mean Ct of the β-actin, an internal control gene.

The total cellular RNA was extracted by Easy Blue^® kits (Intron Biotechnology, Seoul, Korea). From each sample, 500 ng of RNA was reverse-transcribed by TOPscript™ RT Dry MIX, and quantitative real-time PCR amplification was carried out by the incorporation of TB Green^TM Premix Ex Taq (TaKaRa) to detect IL-6, IL-1β, TNF-α, iNOS, COX-2, F4/80, and β-actin mRNA expression. The oligonucleotide primers are listed in Table 4. A dissociation curve analysis of IL-6, IL-1β, TNF-α, iNOS, COX-2, F4/80, and β-actin showed a single peak for each. The mean Ct of the gene of interest was calculated from triplicate measurements and was normalized with the mean Ct of a control gene, β-actin.

Easy Blue^® kits (iNtRON Biotechnology, Seoungnam, Republic of Korea) were used to extract RNA from the colon. A total of 1000 ng RNA was transcribed into cDNA using the ReverTra Ace™ qPCR RT kit (TOYOBO, Osaka, Japan). Real-time PCR was performed using cDNA as a template with QGreen 2X qPCR Master Mix (Cellsafe, Suwon, Republic of Korea). The primer sequences are described in Table 3. Real-time PCR data were normalized using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the housekeeping gene. In this study, real-time PCR was performed using a CFX Connect™ Real-Time PCR System (Bio-Rad, Hercules, CA, USA) using a two-step procedure (40 cycles at 95 °C for 10 s and then at 60 °C for 30 s).

Cellular total RNA was prepared with Easy Blue kits (Intron Biotechnology, Seoul, Korea) under the manufacturer’s instructions. A first-strand cDNA synthesis kit (Amersham Pharmacia Biotech, Oakville, ON, Canada) was used for reverse transcription. We used a SYBR Premix ExTaq™Kit (TaKaRa, Kyoto, Japan) and gene-specific primers. All real-time PCR assays were performed on Thermal Cycler Dice Real Time PCR System (TaKaRa). The National Biotechnology Information Center (NCBI) Primer-BLAST tool was used for primer sequence search. The primer sequences used in the real-time RT-PCR were as follows: for CDH1 sense primer, 5′-GTGCATGAAGGACAGCCTCT, and anti-sense primer, 5′-TGGAAAGCTTCTCACGGCAT; for VIM sense primer, 5′-GGACCAGCTAACCAACGACA, and anti-sense primer, 5′-AAGGTCAAGACGTGCCAGAG; for ACTA2 sense primer, 5′-CCTATCCCCGGGACTAAGACG, and anti-sense primer, 5′-AGAGCCATTGTCACACACCA; for ACTB (β-actin) sense primer, 5′-CCTCGCCTTTGCCGATCC, and anti-sense primer, 5′-CGCGGCGATATCATCATCC. Relative quantification of mRNA expression was carried out using the comparative CT (cycle threshold) method. Mean Ct of the gene of interest was normalized with the mean Ct of a control gene, ACTB (β-actin).

Total cellular RNA was extracted by using Easy Blue^® kits (Intron Biotechnology, Seoul, Korea). RNA (1 μg) was reverse-transcribed (RT) using 0.5 mg/mL random oligonucleotide primers (Promega, Madison, WI, USA) and TOPscript^TM RT DryMIX (Enzynomics, Daejeon, Korea). PCR amplification was performed using the incorporation of SYBR green using SYBR Primix Ex Taq (TaKaRa Bio Inc., Shiga, Japan). The PCR primers used in this study are described in Table S1. Steady-state mRNA levels were determined by real time qPCR using the TaKaRa thermal cycler device. Mean Ct values of genes were calculated from triplicate measurements and normalized versus the mean Ct of GAPDH. The PCR primers used in this study are described in Table S1.