7500c real time pcr detection system

Distal colon total RNA was extracted using an RNA Pure Kit (Carry Helix Biotechnologies Co., Ltd., Beijing, China). The reverse transcription was conducted using a PrimeScript RT Reagent Kit (TaKaRa, Dalian, China). Real-time PCR reactions were performed using SYBR Premix Ex Taq (TaKaRa) under the 7500c Real-time PCR Detection System (Applied Biosystems, Carlsbad, CA, USA). The primers were designed to flank introns with the Primer 5 software (Premier Biosoft, Palo Alto, CA, USA). The primer sets are listed in the Supplemental Table S1. The data were calculated using 2^−ΔΔCT method, normalized to the expression of the housekeeping gene (GAPDH), and expressed as a fold change compared to the normal control group.

Total RNA was extracted according to manufacturer's procedures with the RNA Purification Kit (Aidlab Biotechnologies Co. Ltd., Beijing, China). Total RNA of 800 ng was reverse transcribed to cDNA using PrimeScript RT reagent (Takara, Tokyo, Japan) and diluted 1 : 5 for further experiment. QPCR was performed in a 7500c real-time PCR detection system (Applied Biosystems, Carlsbad, California, USA) using SYBR premix EX Taq (Takara) as described previously [14 (link)]. GAPDH, RPS9, and UXT were used as housekeeping genes for the normalization of other genes' expression. Primers were designed using the National Center for Biotechnology Information (NCBI) Primer-BLAST and listed in Table 1. The 2^−ΔΔCtmethod [15 (link)] was used to calculate the relative mRNA abundance.

RNA was extracted from distal colon using a commercial kit (Carry Helix Biotechnologies Co., Ltd., Beijing, China) and synthesized to cDNA by reverse transcription (TaKaRa, Dalian, China). Quantitative real-time PCR was performed using a two-step amplification method and all information was collected by under the 7500c Real-time PCR Detection System (Applied Biosystems, Carlsbad, CA, United States) and calculated the transcriptional levels of target genes using the 2^–△△Ct method based on cycle threshold (Ct) values and GAPDH was served as the housekeeping gene. Specific primers sequences can be found in our previous published literature (Wang et al., 2016a (link), 2018 (link)).

Total RNA was extracted with the RNA pure Kit (Aidlab Biotechnologies Co., Ltd., Beijing, China) according to the manufacturer's procedures. Reverse transcription of 1 μg total RNA was performed using the PrimeScript RT reagent kit (TaKaRa, Dalian, China). The reverse transcription product was diluted 1 : 10 and used as cDNA template for qPCR analysis. qPCR was carried out in a 7500c real-time PCR detection system (Applied Biosystems, Carlsbad, CA, USA) with the SYBR premix EX Taq (TaKaRa) following the manufacturer's instructions using a standard two-step reaction [25 (link)]. Expression of the housekeeping gene β-actin was used for normalization of other genes' expression. qPCR primers were designed to flank introns with the Primer 5 software (Premier Biosoft International, Palo Alto, CA) and listed in Supplemental Table 1 in Supplementary Material available online at http://dx.doi.org/10.1155/2016/2572175.

According to the instructions from the manufacturer, cellular RNA was extracted using the RNeasy® Plus Mini Kit (Qiagen, Valencia, CA, USA). Reverse transcription was carried out using the PrimeScript RT Reagent Kit (TaKaRa, Dalian, China). Real-time PCR was performed with the 7500c Real-time PCR Detection System (Applied Biosystems, Carlsbad, CA, USA) with SYBR Premix Ex Taq (TaKaRa) following the manufacturer's instructions. Primers were designed to flank introns with the Primer 5 software (Premier Biosoft, Palo Alto, CA, USA), and the primers sets were as follows: GAPDH, 5′-AGGGATGATGTTCTGGAGAG-3′ (F) and 5′-TCAAGATCATCAGCAATGCC-3′ (R); NLRP3, 5′-TCGGAGATTGTGGTTGGG-3′ (F) and 5′-GGGCGTTGTCACTCAGGT-3′ (R); IL-1β, 5′-CTAAACAGATGAAGTGCTCCTTCC-3′ (F) and 5′-CACATAAGCCTCGTTATCCCA-3′ (R); IL-18, 5′-ATCAGGATCCTTTGGCAAGCTTGAATCTAAATTATC-3′ (F) and 5′-ATAGGTCGACTTCGTTTTGAACAGTGAACATTATAG-3′ (R) [23 (link)]; and caspase-1, 5′-TGGTCTTGTGACTTGGAGGA-3′ (F) and 5′-TGGCTTCTTATTGGCACGAT-3′ (R) [24 (link)]. Data were calculated using equation 2^−ΔΔCt and normalized to the expression of the housekeeping gene (GAPDH) and expressed as fold change against the control group.

Total RNA was extracted with the RNA Pure Kit (Aidlab Biotechnologies Co., Ltd., Beijing, China) according to the manufacturer’s instructions. RNA samples were reverse-transcribed using a PrimeScript RT Reagent Kit (TaKaRa, Dalian, China). Quantitative PCR was performed using a 7500c Real-time PCR Detection System (Applied Biosystems, Carlsbad, CA, USA) with SYBR Premix Ex Taq (TaKaRa) following the manufacturer’s instructions. Expression of the housekeeping gene Gapdh was used for the normalization of expression levels. The Caco-2 primers are designed to flank introns with the Primer 5 software (Premier Biosoft, Palo Alto, CA, USA) by our group. Specificity of the primers was checked by the melting curve. The PCR products also have been tested by DNA sequencing and electrophoresed on the agarose gel. The primers sets were as follows: Occludin, 5′-GAGGTTTAGATTAGATTTCCGAC-3′ (F) and 5′-CACAACAAACTCCTTAGAACAAT-3′ (R); ZO-1, 5′-AGATGAACGGGCTACGC-3′ (F) and 5′-GGAGACTGCCATTGCTTG-3′ (R); Gapdh, 5′-AGGGATGATGTTCTGGAGAG-3′ (F) and 5′-TCAAGATCATCAGCAATGCC-3′ (R). Primer sets used in the rat study were obtained from a previous publication [27 (link)].