Lightcycle480 2 real time pcr detection system

One-tenth gram tomato samples were collected in a 2 mL centrifuge tube and frozen in liquid nitrogen. An RNAprep pure plant kit (Tiangen Biotech, Beijing, China) was used for total RNA extraction according to the manufacturer’s instructions. Five-tenths milligram total RNA was used to reverse transcribe to cDNA template by HiScript II Q RT SuperMix for qPCR Kit (Vazyme, Nanjing, China). qRT-PCR was conducted with ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) on a Light Cycle 480 II Real-Time PCR detection system (Roche, Basel, Switzerland). The total 20 μL reaction system was as follows: 10 μL SYBR qPCR Master Mix, 1 μL cDNA template, 10 μM forward and reverse primer, and deionized water. The PCR program was performed with 30 s at 95 °C, followed with 35–40 cycles of 10 s at 95 °C, 30 s at 58 °C, and 1 min at 72 °C. ACTIN was used for calculating the relative expression level of the target gene. The primers are listed in Table S1.

To determine mRNA levels, total RNA was isolated from liver tissue, adipose tissue, 3T3-L1 or BMDM cells by using TRIzol reagent according to manufacturer’s instructions. RNA (2 μg) was reverse transcribed into cDNA using the QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany). Real-time PCR was performed using SYBR Green PCR Master Mix (Invitrogen, Carlsbad, CA, United States) on a CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, United States) or a LightCycle480 II Real-Time PCR Detection System (Roche, Basel, Switzerland). The sequence-specific primers for the genes are listed in Supplementary Table S1. The fold change in expression of each gene was calculated using the 2^−ΔΔCT method, with glyceraldehyde-3-phosphate dehydrogenase as an internal control.