Spin column plant total rna purification kit

After pre-culturing in nutrient solution for 3 weeks, the roots (including 0 to 1 cm and 1 to 2 cm from the root tip) and shoots of HE S. alfredii with 3 replicas were separated. In a separate experiment, another batch of HE and NHE S. alfredii with 3 replicas were exposed to basal nutrient solution with 200 μM ZnSO4 addition or Zn deficiency for 7 d, and the roots were separated. Then, 25 to 50 mg of each part of the plant samples were dried with a paper towel, immediately frozen in liquid nitrogen, and ground into powder. Then, the total RNA of each sample was extracted using a Spin Column Plant Total RNA Purification Kit (Sangon Biotech, Shanghai, China), and converted to single-strand cDNA using HiScript II Q RT SuperMix for qPCR (+gDNA wiper) (Vazyme, Nanjing, China). Gene expression patterns were determined using quantitative real-time PCR with ChamQTM SYBR Color qPCR Master Mix (without Rox) (Vazyme, Nanjing, China). The constitutively expressed SaActin1 gene was used as an internal standard [24 (link)]. The primers designed for the real-time PCR analysis are listed in Table S1. Relative gene expression was calculated using the 2^−∆∆CT method. Sequence data of SaPCR2 can be found in the GenBank under the accession number of HE728063.1 [28 (link)].

Different plant tissues were harvested and immediately frozen using liquid nitrogen. Total RNA was isolated using Spin Column Plant Total RNA Purification Kit (Sangon Biotech, China). The quality and quantity of these RNA samples were further determined using Nanodrop 2000 (Thermo Fisher, US) and agarose gel electrophoresis.

Total RNA was extracted using Spin Column Plant total RNA Purification kit (Sangon Biotech, Shanghai, China) and the integrity and concentration of RNA was determined using agarose gel electrophoresis and Qubit 2.0 Fluorometer, respectively. Library preparation, quality inspection, and sequencing was done as reported earlier [75 ].

The Fungal RNA Kit (Omega, Norcross, GA, USA) and Spin Column Plant Total RNA Purification Kit (Sangon Biotech, Shanghai, China) were used to extract total RNA from T. horrida strains and rice or N. benthamiana leaves, respectively, following the instructions of each kit. The fungal conserved gene UBQ was used as an internal control for data normalization; for rice and N. benthamiana leaves, the Actin gene was used as an internal reference gene to determine the values for the relative expression levels. The cDNA was generated with the Transcriptor First Strand cDNA Synthesis Kit (Roche). The qRT-PCR was conducted on the Bio-Rad CFX96 Real-Time PCR System (Bio-Rad, Foster City, CA, USA) according to the manufacturer’s instructions. We used the 2^–ΔΔCt algorithm to calculate the relative expression levels of target genes [63 (link)]. Four biological replicates were used. Statistical analysis was performed by one-way ANOVA, followed by Tukey’s multiple comparison test. The primer sequences used are listed in Supplementary Table S5.

Total RNA was extracted using a Spin Column Plant total RNA Purification Kit (Order No. B518661; Sangon Biotech, Shanghai, China) according to the manufacturer’s instructions. DNase digestion with Dnase I (Promega, Madison, WI, USA) was performed to remove contaminating DNA. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads (Novogene, Beijing, China) and then broken into short fragments. With these fragments as templates, cDNA were synthesized. To select cDNA fragments of 150–200 bp in length, the library fragments were purified with an AMPure XP system (Beckman Coulter, Beverly, MA, USA). Then, those fragments were selected for PCR amplification as sequencing templates. The PCR products were purified and library quality was assessed on an Agilent Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA). The clustering of the index-coded samples was performed on a cBot Cluster Generation System using a TruSeq PE Cluster Kit v3-cBot-HS (Illumina, Santiago, CA, USA) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina HiSeq 4000 platform (Novogene, Beijing, China) and paired-end reads were generated. Each RNA sample was ligated with a separate adapter and sequenced together in a single run.

Two ecological types of S. alfredii were treated by CdCl₂ with different concentrations (HE: 10 and 100 μM, NHE: 10 μM) and times (0, 6, 24 h, 3, and 7 days). The roots, stems, and leaves of each plant were separated and frozen rapidly in liquid nitrogen. The total RNA was extracted using a Spin Column Plant Total RNA Purification Kit (Sangon) and then synthesized to cDNA with a HiScript II Q RT SuperMix for qPCR (+ gDNA wiper) (Vazyme). Real-time quantitative PCR (RT-qPCR) was performed using a ChamQ SYBR Color qPCR Master Mix (Without ROX) (Vazyme), with LightCycler 480 System (Roche, United States). The RT-qPCR protocol was as follows: 95°C for 3 min, 40 cycles of 95°C for 10 s, 60°C for 30 s. The melting-curve analysis was included to verify the specificity of the primer. The mean amplification efficiency was analyzed with the LinReg software (Ruijter et al., 2009 (link)). The specific primers for RT-qPCR were designed according to the SaPCR2 sequence as follows: SaPCR2 forward 5′-GCGGTGGGATGTGGTCTAC-3′ and SaPCR2 reverse 5′-CGATAATCTCGGCTATTTGGC-3′, SaACTIN1 forward 5′-TGTGCTTTCCCTCTATGCC-3′, and reverse: 5′-CGCTCAGCAGTGGTTGTG-3′ (Chao et al., 2010 (link)). The relative expression levels were calculated using 2^−ΔCt method.

RNA was extracted from 2 g frozen table grape tissue (stored at −80°C) according to the Spin Column Plant Total RNA Purification Kit (Sangon Biotech, Shanghai, China). Both the purity and quantity were checked using a Spectrophotometer (Thermo Scientific, Fresno, CA, United States) at 260 and 280 nm. The concentration and integrity of RNA were evaluated using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, United States). The first-strand cDNA was synthesized from the RNA using the PrimeScript RT reagent kit with gDNA Eraser (Takara-Dalian, China) in PCR System. Specific primers were obtained from Sangon Biotech (Shanghai, China). The RT-qPCR was conducted with Bio-Rad CFX96 Real-Time PCR System (Applied Biosystems, United States) and the computer program set according to Youssef et al. (2020) (link). The RT-qPCR was carried out using T.B. Green^® Fast qPCR Mix (TAKARA BIO Inc., Shiga, Japan) and determined in ABI PRISM 7500 Real-Time PCR System (Applied Biosystems, United States) according to the manufacturer’s instructions. The melting cycle was 95°C for 15 s, 60°C for 1 min, 95°C for 15 s, and 62°C for 15 s. Relative gene expression levels were calculated according to the method of 2^–ΔΔCT (Livak and Schmittgen, 2001 (link)). The experiment was conducted twice, and there were three replications per treatment.