Trnzol universal

The ileum and colon of the mice were homogenized in TRNzol Universal (Tiangen, Beijing, China) to extract total RNA, which was reverse transcribed with ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo, Osaka, Japan). Real-time qPCR was carried out on CFX96 RealTime PCR System (Bio-rad, Hercules, CA, USA). Gene expression was calculated as relative fold changes by the 2^−ΔΔCt method, and β-actin was used as an endogenous control. Specific primers were shown in Table 1.

Total RNA from cells or rat liver tissue was prepared using TRNzol Universal (TIANGEN, Beijing, China) according to the manufacturer’s protocol. Absorbance was measured at 260 and 280 nm to assess the quantity and purity of RNA. The cDNA was prepared from total RNA (1 μg) with a reverse transcriptase (RT) Primer Mix using the PrimeScript RT reagent Kit with gDNA Eraser (TIANGEN, Beijing, China) according to the manufacturer’s instructions. Supplementary Table 1 shows the primer sequences. Subsequent PCR amplification was carried out using a Bio-Rad CFX Manager 3.1 system (Bio-Rad, Hercules, CA) under the following conditions: 40 or 45 cycles at 95 °C for 5 s and at 60 °C for 20 s. Amplified products were monitored by measuring the increase of the dye intensity of the SYBR Green (TIANGEN, Beijing, China) that binds to double-strand DNA amplified by PCR. β-actin was used as an internal control.

Total RNAs of cells were extracted using TRNzol Universal (Tiangen Biotech) according to the manufacturer's guidelines. Then cDNA synthesis was performed using Hifair^® III 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA Digester Plus) (Yeasen Biotechnology). Then qRT‐PCR was performed with QuantStudio5 (Thermo Fisher Scientific) using Hieff^® qPCR SYBR Green Master Mix (Low Rox Plus) (Yeasen Biotechnology) based on the manufacturer's guidelines. We used GAPDH as the endogenous control and the 2^–ΔΔCT method to calculate the gene expression.

We used real‐time fluorescence quantitative PCR (RT‐PCR) technology to detect the expression of related genes. The total RNA was extracted with TRNzol Universal (TIANGEN, Beijing, China). Then, used the FastKing RT Kit With gDNase (TIANGEN, Beijing, China) to purify and reverse transcription. All target mRNAs were amplified by quantitative RT‐PCR using SYBR Green Realtime PCR Master Mix (TOYOBO, Shanghai, China). Table 2 lists the corresponding primers for the genes (VEGF, HIF‐1α, PI3K, AKT and mTOR), all of which are BLAST search design with β‐actin amplification as the control.

CD8⁺ T cells were collected in 1 mL TRNzol Universal (TIANGEN Biotech, DP424), and RNA was extracted with an RNA Simple Total RNA Kit (TIANGEN Biotech, DP419) according to the manufacturer’s protocol. Approximately 200 ng of RNA was reverse-transcribed using the PrimeScript RT Reagent Kit (TaKaRa, RRO37A). The cDNA was diluted 1:10 in RNase/DNAse-free water for quantitative real-time PCR (qRT-PCR) using a CFX Connect System (Bio-Rad). The relative mRNA expression was calculated using 2^−ΔΔCt method. The heat map was described as a fold change, according to the following formula: $Foldchange={log}_2^{2^{-\left(C{t}_{Target}-C{t}_{reference}\right)}}$ . qRT-PCR primer pairs were purchased from Sangon Biotech. Primer sequences are provided in Supplementary Table S4.

Total RNA isolation and RT-qPCR analysis were completed as previously described [29 (link)]. Briefly, total RNA was isolated using TRNzol Universal (TIANGEN, Cat no. DP424). Reverse transcription reactions were performed using FastKing RT Kit (TIANGEN, Cat no. KR116). The qPCR assay was performed under StepOnePlus Real-Time PCR system with Power SYBR Green Master Mix (Applied Biosystems). The qPCR data on qPCR was analyzed by Graph Pad Prism 8. For every gene, each data point was obtained on biological sample triplicate. The relative expression of target genes was normalized by the housekeeper gene Actin1. The related primers are listed in Table S2.

All anther samples from each developmental stage were quick-frozen in liquid nitrogen after collection, followed by rapid transfer and storage in a -80°C freezer for subsequent qRT-PCR experiments. The total RNA of anthers was extracted by TRNzol Universal (Tiangen); cDNA was synthesized by first-strand cDNA synthesis kit (RevertAid Premium Reverse Transcriptase), and the above experiments were carried out according to the kit instructions. Primer premier 5.0 software was used to design the specific primers for qRT−PCR (Quantitative Real-Time PCR). The primer sequence details are provided in Supplementary Table 1. qRT−PCR were performed as previously described (Liu et al., 2021a (link)). The wheat actin gene was used as an internal control and the expression level was calculated by the 2^–⊿⊿Ct method.