Power sybr green

Quantitative real-time PCR was performed to assess the relative quantities of mRNA for IL-6, IL-10, and IL-1β. Total RNA molecules were isolated from frozen tumor tissues using RNA Bee solution (Tet-Test Inc., Friendswood, TX, USA). After quantification of the RNA concentration, the complement DNA (cDNA) was synthesized using a mixture of oligo-dT primer (Invitrogen, Carlsbad, CA, USA), dNTP and reverse transcriptase (Superscript II, Thermo Fisher Scientific, Inc., Waltham, MA, USA). Q-PCR was then conducted using a cDNA template and 2× Power SYBR Green (TOYOBO Co., Osaka, Japan). The reaction cycle at which PCR products exceeded this fluorescence intensity threshold during the exponential phase of PCR amplification was considered as the threshold cycle (CT).

The mRNA levels of PPARγ, C/EBPα, aP2, FAS, adenylyl cyclase (AC), PDE4, CPT1, PPARα, NF‐κB, TNF‐α, IL‐6, and IL‐1β in liver tissues were measured by RT‐qPCR, as previously described (24 (link)). Briefly, total RNA in liver tissues was purified using RNAzol (TelTest Inc., Friendswood, TX, USA) and quantified using a NanoDrop system (Biospecnano, Shimadzu Biotech, Kyoto, Japan), complementary DNA (cDNA) was synthesized using a mixture of total RNA (5 μg), oligo‐dT primer (Invitrogen, Carlsbad, CA, USA), dNTP, and reverse transcriptase (Superscript II, Invitrogen). qPCR was conducted with a cDNA template, 2× Power SYBR Green (Toyobo Co., Osaka, Japan), and specific primers (Supplementary Table S1) using the following cycle: 15 sec at 95°C, 30 sec at 55°C, and 60 sec at 70°C. Fluorescence intensities were determined at the end of the extension phase of each cycle. Values measured during the exponential phase of PCR amplification were used to define threshold cycles (Ct). The expressions of target genes were normalized versus β‐actin (housekeeping gene) based on Ct values at constant fluorescence intensity, as described by (25 (link)).

Total RNA was extracted from frozen mid-colon tissues using the RNA Bee solution (Tet-Test Inc., Friendswood, TX, USA). The RNA concentration was determined, and cDNA was synthesized from the RNA using a mixture of dNTPs, and reverse transcriptase (Superscript II, Thermo Fisher Scientific Inc.), oligo-dT primers (Thermo Fisher Scientific Inc., Waltham, MA, USA). For real-time quantitative PCR (RT-qPCR), the cDNA template was combined with 2× Power SYBR Green (Toyobo Co., Osaka, Japan). The primer sequences employed for mRNA analysis are outlined in Table 1.

RT-qPCR was applied to quantify the relative levels of MUC2, AQP3, and AQP8 mRNAs. After the isolation of total RNAs from mid-colon tissues using RNA Bee solution (Tet-Test Inc., Friendswood, TX, USA), cDNA was synthesized using reverse transcriptase (Superscript II, Thermo Fisher Scientific Inc.). The relative levels of three genes were quantified by the method described in a previous study using 2 × Power SYBR Green (Toyobo Co., Osaka, Japan) [48 (link)]. The primer sequences for the above analyses were as follows: MUC2, sense primer 5′-GCTGC TCATT GAGAA GAACG ATGC-3′, antisense primer 5′-CTCTC CAGGT ACACC ATGTT ACCAG G-3′; AQP3, sense primer 5′-GGTGG TCCTG GTCAT TGGAA-3′, antisense primer 5′-AGTCA CGGGC AGGGT TGA-3′; AQP8, sense primer 5′-CTGAG GCCCT CCCAC ATCT-3′, antisense primer 5′-GGAAA GGAAC AAGGC CAACA-3′; β-actin, sense primer 5′-ACGGC CAGGT CATCA CTATT G-3′, antisense primer 5′-CAAGA AGGAA GGCTG GAAAA GA-3′. The thermal cycling conditions consisted of the holding stage (1 min at 95 °C), cycling stage (40 cycles of 15 s at 95 °C, 15 s at 57 °C, and 45 s at 72 °C), and melt curve stage (15 s at 95 °C and 60 s at 60 °C). Additional analyses, including the fluorescence intensity, the threshold values, the threshold cycle (Ct), and the housekeeping gene, were conducted as described in a previous study [49 (link)].

Frozen bone marrow samples were homogenized using a Polytron PT-MR 3100 D Homogenizer (Kinematica AG, Lusern, Switzerland) in RNA Bee solution (Tet-Test Inc., Friendswood, TX, USA), based on the manufacturer’s instructions. After ethanol precipitation, the total RNAs were harvested by centrifugation at 15,000 rpm for 15 min, after which the concentration was determined by Nano-300 Micro-Spectrophotometer (Allsheng Instruments Co. Ltd., Hangzhou, China). The total complementary DNA (cDNA) against mRNA was synthesized using 200 units of Superscript II reverse transcriptase (Thermo Scientific, Wilmington, DE, USA). RT-qPCR was conducted using the cDNA template obtained (2 μL), along with 2 × Power SYBR Green (10 μL; Toyobo Life Science, Osaka, Japan), and specific primers; Rag2, Forward 5’-TCTGGCCTTCAGTGCCAAAA-3’, Reverse, 5’-CACTGTTAC CATCTGCAGGG-3’. qRT-PCR was progressed for 40 cycles using the following steps: denaturation at 95°C for 15 s, followed by annealing and extension at 70°C for 60 s. The fluorescence intensity of each sample was measured at the end of the extension phase of each cycle. The cycle quantification value (Cq) was defined as described in Livak and Schmittgen’s method [28 (link)].

RT-qPCR was used to measure iNOS, COX-2, tumor necrosis factor (TNF)-α, IL-1β, IL-4, IL-5, IL-6, and IL-10 mRNA levels as previously described [50 (link)]. First, total mRNA was purified from RAW264.7 macrophages and dorsal skin tissues of the subset groups using TRIzol reagent (Favorgen Biotech, Ping-Tung, Taiwan), according to the manufacturer’s protocol. After determining the total RNA concentrations, complementary DNA (cDNA) was synthesized using Superscript II reverse transcriptase (Thermo Fisher Scientific Inc.), and RT-qPCR was performed using the cDNA template (2 μL) and 2× Power SYBR Green (7 μL; Toyobo Life Science, Osaka, Japan) containing specific primers (Supplementary Table S2). RT-qPCR was performed for 40 cycles of denaturation at 95 °C for 15 s, annealing at 70 °C for 60 s, and extension at 70 °C for 60 s. Fluorescence intensities were measured at the end of the extension phase of each cycle. Threshold values for sample fluorescence intensities were set manually and reaction cycles in which the PCR products exceeded these fluorescence intensity thresholds during the exponential phase were considered threshold cycles (Ct). The expression of iNOS, COX-2, TNF-α, IL-1β, IL-4, IL-5, IL-6, and IL-10 was quantified relative to that of the housekeeping gene β-actin, based on a comparison of the Ct values at constant fluorescence intensity [55 (link)].