1st strand cdna synthesis kit

Initially, five upregulated and eight downregulated unigenes were selected to support the accuracy of the RNA-seq results. Total RNA of non-overwintering and overwintering samples were extracted using the methods described above. cDNA was synthesized using 1 μg total RNA template following the instructions outlined by the 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China). The expression levels of 13 genes were determined by qPCR using the Hieff UNICON qPCR SYBR Green Master in the Applied Biosystems 7500 System (USA) according to the manufacturer’s instructions. The PCR procedure was as follows: 5 min at 95°C, 40 cycles of 10 s at 95°C, and 40 s at 60°C, followed by melting curve analysis. Sequence and efficiency of the primer is provided in Supp Table S1 (online only). Relative expression levels were measured based on the 2^−ΔΔCt method with Ribosomal Protein 10 (RPL10) as the internal control for the normalization of data (Li et al. 2018 ). To verify the reliability of RNA-Seq data, Pearson’s correlation between fold changes (Log2 transformed) in RT-qPCR and RNA-Seq results was analyzed.

Total RNA from PBMCs of DVT patients and healthy controls or the experimental cell lines was extracted using the TRIzol Reagent (Invitrogen, Carlsbad, United States). miRNAs and mRNAs were reverse transcribed using the 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China) and PrimeScript RT reagent Kit (Toyobo, Osaka, Japan), respectively, according to the manufacturer’s instructions. The relative expression of each RNA was quantified using SYBR Green (Invitrogen, Carlsbad, United States) based qRT-PCR assay on an Applied Biosystems 7500 instrument (Applied Biosystems, Foster, United States). U6 and GAPDH were used as internal controls for miRNA and mRNA expressional normalization, and relative RNA quantification was calculated via the comparative 2^−ΔΔCt method. For mRNA and miRNA expressional analysis, the PCR primer sequences used are shown in Supplementary Tables S2, S3. Unless otherwise stated, the amplification reaction for each sample was conducted in triplicate.

Total RNAs in cells and tissues were extracted using Trizol reagent (Invitrogen, USA), according to the procedures as previously described [13 (link)]. Subsequently, 1 μg of total RNA was used for reverse-transcription using 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China). Q-PCR was performed using SYBR Green Master Mix (Vazyme, Nanjing, China) on a 7500 Real Time PCR System (Applied Biosystems, CA, USA). The relative gene expression was calculated by the 2-^ΔΔCt method. GAPDH or U6 was used as internal references. The primers used in this study were purchased from Shanghai Shenggong Biological Engineering Co., Ltd. and as follows: circNMD3-forward, 5’-GTTTAATGGAGCTTGAGGGT-3’; reverse 5’-GGTCCATGCACATAAGGAAT-3’. NMD3-forward, 5’-AAGTCTCGATTTCGTTCTGCAA-3’; reverse, 5’-CCTTACTCAGAGGGGCTTTGAT-3’. GAPDH-Forward, 5’-GGAGCGAGATCCCTCCAAAAT-3’; reverse, 5’-GGCTGTTGTCATACTTCTCATGG-3’. miR-498-forward, 5’-TTTCAAGCCAGGGGGCGTTTTTC-3’; reverse, 5’-GCTTCAAGCTCTGGAGGTGCTTTTC-3’. U6-forward, 5’-AACGCTTCACGAATTTGCGT-3’; and reverse, 5’-CTCGCTTCGGCAGCACA-3’.

Total ribonucleic acid was isolated from Nitraria tangutorum leaves with an RNA extraction kit (Bioteke, China) and treated with DNase I (Takara, Japan) to remove contaminating desoxyribonucleic acid. cDNA was synthesized with 1st strand cDNA synthesis Kit (Vazyme, China). The NtSOS2 was amplified from Nitraria tangutorum’s cDNA using specific primers which designed by Oligo 7.60 (Cascade, CO 80809, United States) that were listed in Supplementary Table 1. Homologous proteins were searched for using the NCBI blastp program and multiple alignments were performed using DNAMAN v9.0 (Lynnon Corporation, San Ramon, CA, United States) with the deduced amino acid sequences. A phylogenetic tree was constructed through the Neighbor-Joining method, and the bootstrap value was calculated according to 1,000 repetitions using MEGA-X v10.1 (Temple, Philadelphia, PA, United States).