Abi prism 7500 real time pcr system

Total RNA from Arabidopsis and wheat seedlings was extracted using an RNAprep plant kit (TIANGEN, China). First-strand cDNA was synthesized using a PrimeScript First-Strand cDNA Synthesis kit (TaKaRa, Japan). The qRT-PCR reactions were performed using an ABI Prism 7500 real-time PCR system (ThermoFisher Scientific, USA) using SYBR Green Master Mix (TIANGEN, China) in a total volume of 25 μl and was performed with three technical replications for each sample. A quantitative analysis was performed using the 2-^ΔΔCT method [63 (link)].

NRCM (3.0 × 10⁴ cells/well) were incubated at least 24 h before treatment. Total RNA was isolated with a TRI reagent (Sigma, Burligton, MA, USA) from NRCM treated by S protein for 3 h. cDNA was synthesized with a ReverTra Ace qPCR RT Master Mix kit (Toyobo, Osaka, Japan). Quantitative real-time PCR was performed with ABI prism 7500 Real-Time PCR system (Thermo Fisher Scientific, Waltham, MA, USA) and KAPA SYBR FAST qPCR kit (Roche, Basel, Switzerland). TNF-α primers were forward 5′- AAATGGGCTCCCTCTCATCAGTTC-3′ and reverse 5′- TCTGCTTGGTGGTTTGCTACGAC-3′ [61 (link)]. IL-1β primers were forward 5′-CACCTCTCAAGCAGAGCACAG-3′ and reverse 5′-GGGTTCCATGGTGAAGTCAA-3′ [61 (link)]. IL-6 primers were forward 5′-TCCTACCCCAACTTCCAATGCTC-3′ and reverse 5′-TTGGATGGTCTTGGTCCTTAGCC-3′ [61 (link)]. ACE2 primers were forward 5′- GCAGATGGCTACAACTATAACCG-3′ and reverse 5′-C CCTCCTCACATAGGCATGAAGA-3′. A total of 18 s rRNA amplification were forward 5′-ATTAATCAAGAACGAAAGTCGCAGGT-3′ and reverse 5′-TTTAAGTTTCAGCTTTGCAACCATACT-3′. Data were normalized with 18 s rRNA.

A FlaPure Plant Total RNA Extraction Kit (Jingsha Biology, Beijing, China) was used to extract total RNA from mungbean roots. Reverse transcription synthesis of cDNA was performed using a UnionScript First-strand cDNA Synthesis Mix for qPCR (with dsDNase) (Jinsha Biological, Beijing, China). qRT-PCR was performed with an ABI prism 7500 real-time PCR System (Thermo Fisher, 5823 Newton Dr, Carlsbad, CA 92008, USA) and the reagent used was ChamQ SYBR qPCR Master Mix (Vazyme, Nanjing, China). The procedures used for PCR amplification are referred to in [61 (link)]. The relative expressions of candidate genes from five gene families in mungbeans were analyzed by the 2^−ΔΔCT calculation method. The gene-specific primers were designed by Primer 5 software and the reference gene was VrACTIN3 (EVM0012789) [62 (link)]. Three biological replicates and three technical replicates were set for each group of experiments. Primers are listed in Supplementary Table S1.

Blood samples were collected from each participant, and genomic DNA was extracted using a commercial DNA extraction kit (QIAamp Blood Kit, Qiagen, Valencia, CA, USA). Genotypes for ADIPOQ (rs17366568 and rs182052), RARRES2 (rs17173608 and rs4721), and PPARGC1 (rs8192678 and rs3736265) variants were determined using real-time polymerase chain reaction TaqMan® assays (ThermoFisher, Carlsbad, CA, USA). Amplification was carried out in an ABI Prism 7500 Real-Time PCR System (ThermoFisher). All plates were included negative (all components excluding DNA) and positive internal controls for the genotyping quality conformation. There was 100% consistency in a 30% sample of duplicating test.

According to the manufacturer’s instructions (ThermoFisher, USA, 15,596,026), total RNA was isolated by TRIzol reagent. And 1 μg of total RNA was subjected to reverse transcription (RT) in 20 µl reaction volumes. For the miRNA reverse-transcribed, All-in-One™ miRNA First-Strand cDNA Synthesis Kit (Genecopoeia, USA, QP007) was adopted. In the lncRNA RT reaction, we used PrimeScriptTM RT Master Mix (TAKARA, Japan, RR036A). During the Real-time PCR experiment, Power SYBR PCR Master Mix (Genecopoeia, USA, 4,367,659) was adopted and 2 µL of the cDNA was used as a template. An ABI PRISM 7500 Real-time PCR System (ThermoFisher, USA) was used to conduct amplification reactions, which run with 45 thermocycles of 30s at 94°C, 30s at 55°C, and 30s at 72°C. The primer sequences were listed in Table S1 and S2. Expression levels of each tested gene were determined by the 2^−ΔΔCt method. U6 was used as the internal control for miRNAs, while GAPDH was used as the internal control for lncRNAs.