Lightcycler 480ii real time pcr system

Total RNA was extracted from roots using the Aidlab plant RNA kit (Aidlab Biotech, Beijing, China) based on specifications. The integrity and concentration of the RNA was verified by 1% agarose gel electrophoresis and NanoDrop™ One/OneC (ThermoFisher Scientific, USA). The first strand cDNA was synthesized by PrimerScript RT MasterMix (Takara, Tokyo, Japan) according to the manufacturer’s instructions. qRT-PCR was performed on an LightCycler480 II Real-Time PCR system (Made in Switzerland) using TB Green Premix Ex Taq II (TaKaRa Biotechnology Co. Ltd., Dalian, China) with a 20 μL sample volume. And each reaction mixture contained 2.0 μl of diluted cDNA, 0.8 μl of each primer, 10.0 μl of TB Green Premix Ex Taq II, and 6.4 μl of RNase-free water. qPCR reaction cycling conditions were set as per the manufacturer’s instructions for TB Green Premix Ex Taq II. Each sample was conducted three times biologically using replicate. The relative expression level of each gene was calculated as 2^-ΔΔCT [60 (link)] compared with untreated control plants that were set as 1. Specific primers for CeqMYB genes were designed by Primer Premier 5.0 software and the EF1α was used as housekeeping gene [61 (link)]. Statistical analysis and drawing by GraphPad 8 software [62 ].

To validate RNA-seq data, the expression levels of 12 genes from multiple groups were quantified by quantitative PCR (qPCR) using 2^−ΔΔCt value methods. Ileum total RNA used for RNA-seq was performed to synthesize cDNA using reverse transcriptase Kit (TaKaRa, Dalian, China). Specific primers of these genes were designed using NCBI database, porcine GAPDH gene was designed as an endogenous control (Table S1). The qPCR reactions were performed in 20 μL system involved in 9.5 μL 2 × SYBR Green Realtime PCR Master Mix (TaKaRa, Dalian), 1 μL of forward and reverse primers, 1 μL cDNA and 7.5 μL RNase free ddH₂O using LightCycler 480 II Real-Time PCR System. The cycling conditions included an initial denaturation (95°C for 3 min), followed by 30 cycles (95°C for 15 s; 60 ± 1°C for 15 s; 72°C for 20 s). Three independent biological replicates were performed in triplicate.

Real-time PCR (qRT-PCR) was performed on an LightCycler 480II Real-Time PCR System using SYBR® Green PCR Master Mix (TaKaRa, Dalian, China) with specific primers. Gene expression levels were normalized to beta-actin gene expression through the 2^−ΔΔCt method. Each reaction was performed with a 25 μL reaction mixture that contained 12.5 μL of 2 × SYBR Green Real-time PCR Master Mix, 1 μL of each primer, 2 μL of cDNA, and 8.5 μL of H₂O. PCR cycling conditions were as follows: an initial single cycle at 95 °C for 3 min followed by 40 cycles at 95 °C for 15 s, 57 °C for 15 s, and 72 °C for 20 s. Three independent biological replicates were used for each reaction.

First, total RNA extraction and cDNA synthesis were performed using the RNAprep Pure Plant Kit and FastQuant RT Kit (Tiangen, Beijing, China), respectively. Then, quantitative real-time (qRT)-PCR analysis was performed on a Roche LightCycler 480 II Real-Time PCR system using the PrimeScript RT reagent qPCR Kit (Takara, Dalian, China). All primers were designed by online software Primer-BLAST and displayed in Additional File 1. glyceraldehyde-3-phosphate dehydrogenase (CsGAPDH) was used as housekeeping gene. The relative gene expression level was calculated using the 2^−∆∆Ct method (Livak and Schmittgen, 2001 (link)).

According to the manufacturer's protocol, total RNA was isolated from cell lines by using TRIzol reagent (Invitrogen, USA). Complementary DNA (cDNA) was obtained using a PrimeScript RT reagent kit (TaKaRa, Japan). Real-time PCR was performed using TB Green Premix Ex Taq II (TaKaRa, Japan) in a Light Cycler 480 II Real-Time PCR system. Glyceraldehyde phosphate dehydrogenase (GAPDH) was employed as a control for normalization. The primers were shown as follows: FH forward 5′-CCGCTGAAGTAAACCAGGATTATG-3′ and FH reverse 5′-ATCCAGTCTGCCATACCACGAG-3′; and GAPDH forward 5′-GTCTCCTCTGACTTCAACAGCG-3′ and GAPDH reverse 5′-ACCACCCTGTTGCTGTAGCCAA-3.