Rna isolation kit

Isolation of RNA from the leaves (same leaves used for protein analysis) was performed using an RNA isolation kit according to the manufacturer’s instructions (Promega, Madison, WI, USA). One μg of DNAase-treated RNA was reverse transcribed using a reverse transcriptase kit (Promega, Madison, WI, USA) to synthesize first-strand cDNA. Quantitative Real-time PCR was performed with a Rotor-Gene Q 2plex HRM Platform (Rotor-Gene Q 2plex HRM Platform), using SYBR green as a reference dye provide by Qiagen qPCR kit (QIAGEN OneStep RT-PCR Kit, Westburg, Netherland) for 5 min at 95°C, followed by 25 cycles consisting of 20 sec at 95°C, 30 sec at 57°C and 30 sec at 72°C, then 10 min at 72°C. All quantifications were normalized to actin. The RT-PCR reactions were performed using three independent RNA preparations from independently grown plants and three replicates for qPCR. The gene specific primers used in this study are as follows: lePT1 (accession number: AF022873.1) F-5’-ATAAAAATGCAAAATAATCC-3’; R-5’-AGCCACCCGAAGAACAACTG-3’; lePT2 (accession number: AF022874.1) F-5’-AGAAAGTGCACAATTTTTTG-3’; R-5’-GGTGTACTACCAAAGGAGAG-3’; leActin (accession no. U60482) F-5’-CTGCCATGTATGTTGCCATC-3’; R-5’-GGCTGTGGTGGTGAAAGAGT-3’.

The leaves, tendrils, flower buds, flowers and fruits were frozen in liquid nitrogen and stored at −80°C prior to RNA extraction. Total RNA was extracted with Huayueyang RNA isolation kit (China), cDNA was synthetized using M-MLV Reverse Transcriptase (Promega). Quantitative RT-PCR was performed using an Applied Biosystems 7500 real-time PCR systems with SYBR Green as fluorescent dyes (TaKaRa). Three biological replicates were performed, upon which three technical replicates were used for the qRT-PCR analysis. 18S rRNA was used as reference control to normalize the expression data [36] (link). The gene specific primers are listed in Table S1.

Inflorescence of sup-5, pAtSUP::CsSUP;sup-5, 35S::CsSUP;sup-5 were frozen in liquid nitrogen and stored at −80°C prior to RNA extraction. Total RNA was extracted with Huayueyang RNA isolation kit (China), cDNA was synthetized using M-MLV Reverse Transcriptase (Promega). Actin2 was used as internal control to normalize the expression data. The gene specific primers are listed in Table S1.

Total RNA was extracted from female flower buds using the Huayueyang RNA isolation kit (China), and 3 µg of samples were used to synthetize cDNA using M-MLV Reverse Transcriptase (Promega). The cDNA samples were amplified by PCR via the following system: 95°C for 5 min; 30 cycles of 95°C for 30 s, 53°C for 30 s, and 72°C for 30 s; then 72°C for 10 min.

The analysis of qRT-PCR was performed following the protocol that was formerly described by Nie et al. (2016) (link) with minor modification. Spores were inoculated with corresponding medium (PDA and YES agar for conidiation, WKM media for sclerotia formation) for 48 h, and the fungal hyphae were collected and ground into powder in liquid nitrogen. Then, total RNA was extracted with RNA isolation kit (Promega, United States). The first strand cDNA was synthesized with Revert Aid First-strand cDNA Synthesis Kit (TransGen Biotech, China) from 3 μg total RNA. The primers for the analysis were listed in Supplementary Table S2. Each experiment was repeated at least five times.

LBPs (≥90%) were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Potassium oxonate (97%) and allopurinol (≥99%) was purchased from Sigma-Aldrich Co. LLC (Shanghai, China). Uric acid (UA), serum creatinine (S_CR), blood urine nitrogen (BUN) and xanthine oxidase activity assay kit were purchased from Abcam Inc. (Cambridge, UK). The RNA isolation kit was obtained from Promega Biotechnology Co., Ltd. (Beijing, China). PrimeScript^TM RT reagent kit with gDNA Eraser and TB Green^® Premix Ex Taq™ II (Tli RNaseH Plus) was purchased from Takara Biomedical Technology Co., Ltd. (Dalian, China). Protease and phosphatase inhibitors were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Anti-OAT1, anti-OAT3 and β-actin antibody were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Anti-GLUT9 was purchased from Abcam Inc. (Cambridge, UK). The Amersham ECL System was purchased from GE Healthcare (Pittsburgh, PA, USA). Other biochemical reagents and chemicals were of analytical grade.