Hiscript 2 reverse transcriptase

Hippocampal tissues were isolated from the brains of both control and surgery mice on day 1 post-surgery. Total RNA was extracted from hippocampal tissues using TRIzol (Invitrogen) and subjected to DNase I digestion for genomic DNA removal (Raihan et al., 2019 (link)). Reverse transcription was conducted using HiScript II Reverse Transcriptase (Vazyme) following the instructions of the manufacturer. Quantitative polymerase chain reaction (qPCR) was performed with AceQ qPCR SYBR Green Master Mix (Vazyme) on a Light Cycler 96 (Roche) instrument according to standard procedures. The real-time value for each sample was averaged and compared using the CT method, where the amount of target RNA (2^–ΔΔCT) was normalized to a reference (ΔCT). The relative levels in the surgery group over the control group were plotted accordingly. qPCR detection primers are listed in Table 1.

The total RNA was extracted from the frozen Arabidopsis leaf samples with RNAiso Plus (Takara 9101). The resulting RNA samples were treated with DNase I (TransGen Biotech GD201-01) followed by reverse transcription using HiScript II Reverse Transcriptase (Vazyme R223-01) according to the manufacturer’s instructions. qRT-PCR was performed in triplicate using SYBR Green Real-Time PCR Master Mix (Vazyme Q711-02) on the Bio-Rad CFX96 system. The 2^{− ∆∆ Ct} method was used to calculate the relative gene expression level across the samples. Finally, the results were presented as histograms by GraphPad Prism 8 software (GraphPad, San Diego, CA, USA). Primers used are listed in Table S3.

Sixteen candidate genes involved in glycolysis, the tricarboxylic acid cycle (TCA) and amino acid anabolism were chosen and RT-qPCR was conducted to verify the gene expression. The specific primers were designed using Primer Premier 5.0 (Table S2). Total RNA was reverse-transcribed using HiScript II reverse transcriptase according to the manufacturer’s instructions (Vazyme, Nanjing, China). Real-time PCR experiments were performed using a CFX Connect Real-Time PCR system (Bio-Rad Laboratories Inc., Hercules, CA, USA). β-Actin was used as the internal standard gene (Zhao et al., 2018 (link); Lin et al., 2021 (link)). The relative expression of genes was calculated by the 2^−∆∆Ct method (Salvatierra et al., 2010 (link)). All RT-qPCRs were performed with three biological replicates. All statistical analyses were conducted by SPSS 17.0 software (SPSS Inc. Chicago, IL USA). The data are displayed as the means ± standard deviations (SD).

The hepatic total RNA of six fish in each group was extracted using Trizol Reagent (TaKaRa, Japan). The quantity and the integrity of RNA were determined by an Eppendorf BioPhotometer and agarose gel electrophoresis method (Hamburg, Germany). The hepatic cDNAs of grass carp were achieved from 1 μg total RNA according to the instructions of HiScript® II Reverse Transcriptase (Vazyme Biotech, China).

The expression levels of eight selected genes were determined using real-time quantitative PCR (RT-qPCR) to verify the expression patterns of the mRNAs identified by RNA-seq. RT-qPCR was performed on CFX Connect (Bio-Rad Laboratories Inc. Hercules, CA, United States) using HiScript II reverse transcriptase (Vazyme Biotech Co. Ltd., Nanjing, China), according to the manufacturer’s protocol. The primers used for RT-qPCR are shown in Supplementary Table 3. Sample cycle threshold (Ct) values were determined and normalized against the constitutively expressed gene (β-actin-1, internal control) (Zhao et al., 2018 (link)). The 2^–ΔΔCT method was used to calculate the relative gene expression level. Three biological replicates and three technical replicates were used for all RT-qPCR reactions.

Approximately 100 mg mycelia was ground in liquid nitrogen, and total RNA was extracted using a phenol/SDS method (Zhang et al., 2018 (link)). RNA integrity and quantity were checked using an Agilent 2100 Bioanalyzer (Agilent Technologies, CA, United States). The cDNA strand was synthesized using HiScript II Reverse Transcriptase (Vazyme, Nanjing, China) with 500 ng total RNA in a 20-μl reaction according to the manufacturer’s instructions. The primer sequences for the tested genes and the reference gene are listed in Supplementary Table 1. The quantitative RT-PCR was performed using a CFX Connect real-time PCR system (Bio-Rad). Each reaction contained 1 μl each of forward and reverse primer (10 mM), 30 ng sample cDNA, 10 μl AceQ qPCR SYBRMaster Mix (Vazyme), and ddH₂O to a 20-μl final volume. A two-step RT-PCR protocol was adopted for all amplifications and comprised a denaturation step at 95°C for 2 min, followed by 40 cycles of 95°C for 15 s and 60°C for 20 s, with the fluorescence measured at the end of each cycle. A dissociation curve was generated to verify that a single product was amplified. Transcript levels were normalized with the internal reference gene INIF2 and quantified according to Zhang et al. (2018) (link). Three biological and three technical replicates were analyzed.

Total RNA was extracted using the TRIzol. cDNA was reversed transcribed by HiScript II Reverse Transcriptase (Vazyme biotech R222-01) from 2 μg of total RNA. Real-time quantitative PCR was performed with ChamQ^TM SYBR^® qPCR Master Mix (Vazyme biotech Q311-02). Relative expression values were normalized to Gapdh and analyzed with Microsoft Excel 2019. Each experiment was operated in technical triplicate. Primers for RT-qPCR were listed in Supplementary Data 1.