Total rna kit

The total RNA of HUVECs was extracted using the total RNA kit (TaKaRa, Japan), followed by the manufacturer’s instructions. The concentration and purity of the extracted RNA were determined using Nanodrop One (Thermo Fisher, United States). The integrity and contamination of RNA were evaluated by agarose gel electrophoresis (Bio-RAD, United States). And the integrity of the RNA was further verified using an Agilent 2100 bioanalyzer (Agilent 2100, United States). The preparation of specific transcriptome libraries was performed by removing ribosomal RNA. RNA was degraded using Ribonuclease (RNase) H (TaKaRa, Japan). The first single-stranded cDNA was synthesized with reverse transcription of RNA, followed by the second double-stranded cDNA synthesized using dNTP (dUTP, dATP, dGTP, and dCTP). Subsequently, RT-qPCR was performed to amplify the sequences and then the preparation of the total RNA library was completed. The RT-qPCR and the Agilent 2100 bioanalyzer were used to control the quality and quantity of the library. Transcriptome sequencing was performed with Illumina PE150.

Based on the results of transcriptome analysis, six candidate genes were selected, the corresponding nucleotide sequences were searched in the transcriptome, and the selected DEGs were verified by qRT-PCR. The total RNA was isolated from S16-CK, S16-HS, J7-CK, and J7-HS using a total RNA kit (Takara Biomedical Technology Co., Beijing, China). Primer 6 software was used to design specific primers for the six genes identified from the transcriptomic data. The qRT-PCR primers designed for the selected DEGs genes are listed in Table S6; the gene encoding actin was used as an internal reference gene. qRT-PCR was performed using SYBR GREEN Master Mix (Vazyme Biotechnology Co., Ltd., Nanjing, China). Relative gene expression levels were calculated using the 2^−ΔΔCT method [61 (link)].

Total RNA was extracted from renal cortical tissue and left/right ventricular free wall using Total RNA kit (Takara, Japan) according to the manufacturer’s instructions. Reverse transcription and cDNA synthesis were accomplished using RNA PCR kit (Takara, Japan). Real-time polymerase chain reaction was performed to detect the expression of various cytokines by Step One SYBR Green Mix Kit (Takara, Japan) and ABI Prism Sequence Detection System (Applied Biosystems, USA) according to the manufacturer’s instructions. The conditions of amplification reaction were 95 °C for 30 sec, 95 °C for 5 sec, 60 °C for 30 sec, and PCR was done for 40 cycles. PCR primers are shown in Table 3. Relative gene expression was calculated using the 2^−ΔΔCT method.

Total RNA from the leaves was extracted using the total RNA kit (TaKaRa, Dalian, China). The Two-Step Prime-Script TM RT Reagent Kit (Perfect Real Time; TaKaRa) with gDNA Eraser was used for the reverse transcription reactions following the manufacturer’s instructions. Twelve DAPs were randomly selected for RNA-level examination. The specific primers were designed using Primer 5.0 (Premier Biosoft, Palo Alto, CA, USA). The cDNA samples were used as templates and mixed with primers and SYBR Premix ExTaq (TaKaRa) for real-time PCR analysis. Real-time PCR was conducted using a BioRad IQ5 Real-time PCR Detection System (Bio-Rad, Hercules, CA, USA). The temperature settings were 95 °C for 5 min followed by 40 cycles of 95 °C for 15 s, 60 °C for 15 s, and 72 °C for 15 s. TaActin and TaGAPDH were used as reference genes to normalize the expression level of target genes, respectively. Relative gene expression was computed using the 2^−ΔΔCt method [73 (link)]. All primers used in this study are listed in Table S6.

For the total RNA extraction, the frozen K. obovata leaves (0.1g) were ground in liquid nitrogen and extracted by using a Total RNA Kit (TaKaRa, Dalian, China). We observed the RNA quality and integrity with an ultraviolet spectrophotometer (Cary 50, Varian, USA) and agarose gel electrophoresis. The RNA was used to synthesized cDNAs with moloney murine leukemia virus (M-MLV) reverse transcriptase First-Strand cDNA synthesis kit (TaKaRa, Dalian, China), and the cDNA mixture was used as a template for subsequent PCRs. The primers used for real-time PCR are shown in Supplementary Table S4. A 10 μL real-time PCR mixture contained 2 μL of primers, 2 μL of cDNA, and 6 μL of SYBR Green (Sangon, Shanghai, China). Five independent biological replicates were used to perform gene expression. The relative gene expression was calculated by the 2^−ΔΔCT method, and actin was used as an an internal control [74 (link)]. The Bio-Rad iQ5 Multicolor Real-Time PCR Detection System (Bio-Rad, Hercules, CA) was used to run qRT-PCR.