Primescript rt reagent kit with gdna erase

Total RNA was extracted from SlGRXC6-scilenced-, SlNTRC80-scilenced-, or SlGRXC6-overexpressed tomato leaves at different time points using Trizol Reagents (Life Technologies, USA). RNA samples were treated with DNase I and converted to cDNA following manufacturer’s instructions (PrimeScript RT reagent Kit with gDNA Erase, Takara, Japan).
To quantify viral DNA levels by qPCR, total DNA was extracted from mock- or TYLCV-infiltrated tomato leaves at different time points and TYLCV V1 was detected to represent the viral genomic DNA. The qRCR reaction mixes consisted of 6 μl of SYBR Green supermix (Bio-Rad, USA), 0.10 μl of each primer (10 pmol) and 1.5 μl of DNA or cDNA (10 ng/μl) in a total volume of 12 μl. SlActin was used as an internal control for tomato. Each experiment was performed in triplicate and repeated three times. PCR reactions were done in an Applied Biosystems 7500 (ThermoFisher Scietific, USA) real-time PCR detection system. Data analysis was performed using Applied Biosystems 7500 software version 2.0.6.

Total RNA of fruit peel was extracted using a modified CTAB method according to Zhang et al.'s protocol (2013). First‐strand cDNA was synthesized from 4 μg DNA‐free RNA using the oligo (dT) PrimeScript™ RT reagent Kit with gDNA Erase (RR047Q, TaKaRa, Otsu, Japan) following the manufacturer's instructions. The cDNA was diluted threefold and used as the template for gene cloning and qPCR analysis. qPCR was performed using a CFX96 real‐time PCR detection system (CFX96, Bio‐Rad, Hercules, CA, USA). A template‐free negative control for each primer pair was included for each run. All relative expression levels were calculated using the 2−ΔΔCt method against the pear Actin or Arabidopsis PP2A (JN684184 or AT1G13320, respectively) gene. The analysis was performed with three biological replicates. The primers were designed using the Primer‐BLAST tool (http://www.ncbi.nlm.nih.gov/tools/primer-blast/). The amplification efficiencies and product specificities of the primers were determined using standard curves and PCR‐product sequencing. The primers used for qPCR are listed in Table S1.

Total RNA from SH-SY5Y cells was extracted using RNAiso Plus (Takara). The PCR forward and reverse primers for amplifying GAPDH were 5′-AGAAGGCTGGGGCTCATTTG-3′ and 5′ -AGGGGCCATCCACAGTCTTC-3′, respectively. Real-time RT-PCR was performed using the primeScript RT reagent kit with gDNA Erase and SYBR Premix Ex Taq kit (Takara) in a CFX9 Real-Time PCR Detection System (BioRad, Hercules, CA). The products were treated with 300 µM CrO₃ or 150 µM CrCl₃. Melting of DNA was performed from 60 to 95°C at 0.1°C/s with a smooth curve setting. The melting peaks were visualized by plotting the first derivative against the melting temperature. A melting temperature (Tm) was defined as the peak of the curve.

To investigate the tissue distribution of AdPGRP-S1, ten tissues including liver, spleen, intestine, muscle, brain, stomach, kidney, lung, heart and skin were collected from four normal salamanders. Total RNA was extracted from these tissues using Tirzol reagent (Invitrogen, USA), and reverse transcribed into first strand cDNA using PrimeScript^® RT reagent Kit with gDNA Erase (Takara, Japan). Real-time qPCR was performed using SYBR Green fluorescent dye (Invitrogen, USA) on the CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad, USA) and analyzed as described previously [43 , 44 (link)]. The expression level of AdPGRP-S1 was normalized to that of β-actin. Primers used for real-time quantitative PCR are listed in Table 1.

As the described method in the previous report (25 (link)) with slightly modification. Fecal DNA was extracted using the Stool Genomic DNA Extraction Kit (Applied by TIANGEN, Beijing, China). And the intestinal tissue homogenized in Trizol reagent (Beyotime, Jiangsu, China) and total RNA was isolated, and RNA from each sample was reversely transcribed into complementary DNA by PrimeScript™ RT reagent Kit with gDNA Erase (Takara). Real time-PCR was performed using the TB Green^® Premix Ex Taq™ (TaKaRa) by following the manufacturer's instructions. Real time-PCR was performed using CFX96™Real-Time System (Applied Biosystems). The primers for Real time-PCR were designed and synthesized (Sangon Biotech, Shanghai, China) and the sequence information was shown in Table 2. Results were calculated using the 2^−Ct method.

To validate the sequencing data, we first selected nine DEGs for qRT-PCR. Total RNA was extracted from the leaf tissues of YX and YX-yl. The PrimeScript™ RT reagent Kit with gDNA Erase (Takara, Dalian, China) was used to synthesize first-strand cDNA. qRT-PCR was performed using gene-specific primers in a total volume of 10 μL comprising 5 μL of SYBR Premix Ex Taq II, 3 μL of ddH₂O, 1 μL of primer mix (1:1 mix of forward and reverse primers at 10 μmol/μL each), and 1 μL of cDNA as a template. The reaction conditions were as follows: 30 s at 95 °C, followed by 39 cycles of 5 s at 95 °C, 30 s at the annealing temperature, and 5 s at 60 °C to 95 °C. The primers used for qRT-PCR analysis are listed in Table S6. The actin gene was used as an internal standard. Relative expression levels were calculated as 2^−△△ct [53 (link)].