Abi 7500 real time quantitative pcr system

Total RNA was extracted from ileum tissues with TRIzol reagent (Takara Biotechnology, Dalian, China) according to the manufacturer’s instructions. The quantity and quality of the RNA were measured with a Nanodrop 2000 (NanoDrop Technologies, Wilmington, DE, USA) instrument and verified by electrophoresis on a 1.5% agarose gel. Total RNA (0.5 mg) was reverse-transcribed with random primers according to the manufacturer’s protocol (Takara Biotechnology). The resulting complementary DNA was diluted and used as a PCR template to evaluate gene expression. Quantitative RT-PCR was conducted on an ABI7500 Real-Time Quantitative PCR System (Thermo fisher, Waltham, USA) using SYBR Premix Ex Taq kits (Takara Biotechnology) under the following conditions: pre-denaturation at 95 °C for 30 s and 40 cycles of 95 °C for 5 s, 60 °C for 30 s, and 72 °C for 15 s. A dissociation curve was constructed at the end of the reaction to ensure that only one amplicon was amplified. Primers for the genes of interest were designed on the basis of the pig (Sus scrofa) sequence (Supplemental Table S1). All experiments were repeated in triplicate, and β-actin was used as an internal control for normalization. All mRNA relative expression levels were calculated with the comparative Ct method (Livak and Schmittgen 2001 (link)).

Part of the spleen cells was lysed with TRIzol reagent (Takara, Dalian, China) and stored at -80°C for total RNA extraction. cDNA was synthesized by reverse transcription (RT) using the PrimeScript RT kit (Takara, Dalian, China). The relative expression levels of Tnf, Il1b, Il6, Il10, Il17, Ifng, Tbet, and Gata3 genes in mice spleen cells were detected by real-time quantitative polymerase chain reaction (RT-qPCR). RT-qPCR was performed using the ABI 7500 real-time quantitative PCR system (Applied Biosystems, Inc., Foster City, California) under the following conditions: 95°C reaction 30 seconds, 95°C reaction 5 s, and 63°C reaction 34 s, 45 times, and then, the reaction at 95°C for 15 s, 60°C for 1 min, 95°C for 15 s, and 60°C for 15 s, once. GAPDH was an internal reference gene. The mRNA relative expression levels of target genes were calculated and detected by the 2^-ΔΔCT method. Table 1 shows the primer sequences.

The skin samples were extracted with Trizol reagent (Invitrogen, Carlsbad, CA, United States) to obtain the total RNA. Then the RNAs were reverse transcribed into cDNA with M-MLV reverse transcriptase (Promega, Madison, United States). Quantitative PCR was performed with SYBR Green qPCR Supermix (Invitrogen, Carlsbad, CA, United States) on an ABI 7500 real-time quantitative PCR system (Applied Biosystems, Foster City, United States). The PCR cycle parameters were as follows: denature at 95°C for 5 min, followed by 40 cycles of 95°C for 15 s (denature) and 60°C for 32 s (annealing). Data were analyzed with the 2^−ΔΔCT method. Primers used were listed in Table 2.

Using a Plant RNA Purification Reagent (Invitrogen), total RNA from fruit at various growth and ripening phases was isolated in according with the manufacturer’s instructions. The DNase (TAKARA, Dalian, China) enzyme was used to degrade the genomic DNA. The Hifair II 1st Strand cDNA Synthesis SuperMix for RT-qPCR (YEASEN, Shanghai, China) was used to create the cDNA and SYBR Green (Vazyme, Nanjing, China) was used in the RT-qPCR amplification processes using an ABI 7500 Real-Time quantitative PCR System (Applied Biosystems, United States). The grape Actin gene was used as an internal control in the analysis of three biological replicates. The primers for RT-qPCR are listed in Supplementary Table S8.