Hiscript first strand cdna synthesis kit

Total RNA was extracted in the same way as for the microarray analysis and cDNA was synthesized using the Hiscript First-strand cDNA Synthesis Kit (Vazyme, Nanjing, China) according to the manufacturer’s instructions. qPCR assay was performed using ABI 7500 Real-Time PCR System (Applied Biosystems, American). Primers used in this study were as follows: MMP1 forward, 5′-GGGAGATCATCGGGACAACTC-3′, and reverse, 5′-GGGCCTGGTTGAAAAGCA T-3′; MMP2 forward 5′-CCGTCGCCCAT CATCAAGTT-3′, and reverse, 5′-CTGTCTGGGGCAGTCCAAAG-3′; GAPDH forward, 5′-GCACCGTCAAGGCTGAGAAC-3′, and reverse, 5′-TGGTGAAGACGCC AGTGGA-3′. The relative levels of the target mRNA normalized to GAPDH mRNA were evaluated by 2^-ΔΔC method.

The total RNA was extracted using the TransZol Up Plus RNA Kit (Transgen, Beijing, China) from the leaves of the rice seedlings, and ≥1 μg of total RNA was reverse transcribed using a HiScript First-Strand cDNA Synthesis Kit (Vazyme, Nanjing, China). To detect the expression levels of genes, qRT-PCR was performed with Hieff qPCR SYBR Green Master Mix (YEASEN, Shanghai, China). Data were standardized to the internal reference gene Actin1, and relative quantification was used for data analysis. Three biological replicates were performed in each experiment.

The replication of DHAV-1 in DEF cells was determined by an RT-qPCR assay. For establishment of standard curve, reverse-transcription (RT)–PCR assay was performed to amplify a 214-bp cDNA fragment from the 5′UTR region of the C80 genome, employing previously reported reactions, conditions [35 (link)], and the qDHAV-1-f/r primer sets (Table S1) [36 ]. The PCR product was cloned into the pCloneEZ-Blunt-AMP/HC (Taihegene, Beijing, China) to construct recombinant plasmid pC-C80. Then, 2 μL from each of 10-fold dilutions (10⁻³–10⁻⁷; corresponding to 4.39 × 10⁵–43.9 copies/μL for pC-C80) of the recombinant plasmid was employed to generate standard curve with an AceQ qPCR SYBR Green Master Mix (Vazyme, Nanjing, China), according to the manufacturer’s instructions. Amplification was performed on an Applied Biosystems StepOne™ Real-Time PCR System (Thermo Scientific, Waltham, MA, USA), with the cycling conditions provided by the AceQ qPCR SYBR Green Master Mix. For virus quantification, RNA was extracted from 200 μL of each sample, using a TRIzol reagent (Thermo Scientific, Waltham, MA, USA) and dissolved in 50 μL of RNase-free water. Then, 5 μL of RNA was reverse transcribed into cDNA, using a HiScript First Strand cDNA Synthesis Kit and random hexamers (Vazyme, Nanjing, China). Then, 2 μL of cDNA was used for the determination of viral load. Each sample was detected in triplicate.

TRIzol (Invitrogen) was used to extract the RNA and the instructions of the HiScript First Strand cDNA synthesis kit (Vazyme Biotech Co., Ltd., Nanjing, China) were followed to create the cDNA. Real-time (RT) PCR analysis was conducted according to the method described in the UltraSYBR one-step RT-qPCR kit (CWBIO, Beijing, China). In Table S1, the primers used for RT-PCR analysis were mentioned.

Whole blood of 10 patients (IDs: 19010101, 19010102, 19010104, 190101010, 19010111, LXY, DTY, LC, OYZY, and GJX) and 10 healthy controls were first processed with Red cell lysis buffer (Sangon Biotech, Shanghai, China) and then treated with TRIzol (Invitrogen, Carlsbad, CA, USA) to extract total RNA. Reverse transcription was performed with HiScript First Strand cDNA Synthesis Kit (Vazyme, Nanjing, China) to obtain cDNAs. Then qPCR was performed on Bio-Rad CFX96 (Bio-Rad Laboratories Inc., Hercules, CA, USA) with AceQ qPCR SYBR Green Master Mix (without ROX) (Vazyme, Nanjing, China) according to the manufacturers’ protocols. ACTB gene was used as the reference, and the primer sequences are listed in Supplementary Table S1. Cycling conditions were as follows: 95°C for 5 min, followed by 40 cycles of 95°C for 10 s and 60°C for 25 s.