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46 protocols using Evo M-MLV RT Mix Kit

DMEM medium supplemented containing 10% fetal bovine serum (FBS) was used to maintain A375 and HaCaT cell lines. These cells were cultured in a humidified chamber at 37°C with 5% CO2. The extraction of total RNA from cells was done by using a Trizol reagent (Sangon Biotech, China) according to the instructions supplied by the manufacturer. Ultraviolet absorption spectrometry was used to assess RNA quantity and quality. Evo M-MLV RT Mix Kit (Accurate Biotechnology, Hunan, China) from total RNA (1 μg) was used for cDNA synthesis, Evo M-MLV RT Mix Kit (Accurate Biotechnology, Hunan, China) from total RNA (1 μg) was used. Quantitative PCR (qPCR) was performed using the SYBR Green Premix Pro Taq HS qPCR Kit (AG11718, Accurate Biotechnology, Hunan, China). Primers shown in Table S3 were used for amplifying products. The ΔΔcycle threshold method was used for quantifying the relative gene expression. GADPH acted as an endogenous control to normalize the expression level of relative gene expression. All results were obtained from at least three independent experiments with a minimum of three replicates per condition.
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We used 56 flavonoid-related genes in A. thaliana to search for homologs in the ZS11 genome at BnPIR [66 (link)]. Newly assembled genes, whose protein sequences had best hits with the flavonoid-related genes in A. thaliana using BLASTP, were also included. The protein sequences of A. thaliana were downloaded from https://www.arabidopsis.org/. The integrative software TBtools (v1.108) [67 (link)] was used to illustrate the distribution of the flavonoid-related genes on the chromosomes of ZS11. DEGs were chosen for the validation of the RNA-seq data using qRT-PCR. Gene-specific primers were designed using Primer v3.0. All the genes, primers, and sizes of the amplicons are listed in (Supplementary Table S9). The subsamples for the RNA-Seq were first reverse transcribed into cDNA using an Evo M-MLV RT Mix Kit (Accurate Biotechnology, Changsha, China), according to the manufacturer’s protocol, and SYBRqPCR Master Mix (Vazyme) was used for a real-time qPCR on a Bio-Rad CFX-96 Real Time PCR System (Bio-Rad). The relative expression levels were analyzed using the –ΔΔCt method, with BnaC02g00690D (ACT7) used as an internal reference. Each sample included three biological replicates.
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Total RNA was isolated using TRIzol (Invitrogen, CA, USA,15596026), and the resultant RNA purity and concentration were measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific, MA, USA, ND2000). Briefly, an Evo M-MLV RT Mix Kit (Accurate Biotechnology, Hunan, China, AG11705) was used to synthesize complementary DNA. miRNA was reverse transcribed into complementary DNA using the miRNA 1st strand complementary DNA synthesis kit (Accurate Biotechnology, Hunan, China, AG11717). A SYBR® Green Premix Pro Taq HS qPCR Kit (Accurate Biotechnology, Hunan, China, AG11701) was used to perform qRT-PCR on a Bio-Rad CFX Real-time PCR system (Bio-Rad, CA, USA). β-Actin and U6 were used as internal references for mRNA and miRNA transcript-levels quantification, respectively. Absolute qRT-PCR was performed as reported previously (35 (link)). Standard curves of mir222hg and miR146a-5p were generated via the serial dilution of standard mir222hg (Tsingke Biotechnology, Beijing, China) and standard miR146a-5p (Accurate Biotechnology, Hunan, China). Data are presented as copies per 100 pg total RNA (2×105 cells). The primers were synthesized by Sangon Biotech (Shanghai, China) (32 (link)). The primer sequences for all genes are provided in Table 3.
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Another half of the samples mentioned above were cut into pieces, and ground with small steel balls in a cold mill. Total RNA was isolated from the samples by using Trizol (Invitrogen) according to the manufacturer’s instructions. Reverse transcription from total RNA (1000 ng) was performed using Evo M-MLV RT Mix Kit (AG11728, Accurate Biotechnology, Hunan, Co., Ltd) with gDNA Clean for qPCR. RT-PCR reactions were carried out using the SYBR Green PCR Kit (AG11701, Accurate Biotechnology, Hunan, Co., Ltd). After a 30-s predegeneration at 95°C, 40 cycles were performed: denaturation at 95 seconds for 15 seconds, extension at 60°C for 30 seconds followed by a 65 to 95°C solubility curve, which was constructed to analyze the fluorescence signal. The relative amount of mRNA was normalized against GAPDH (Supplementary Information 1). The primers (Table 1) were produced by Shanghai Sangong Biotechnology (Shanghai, China). The relative expression levels were determined by the 2−ΔΔCT method and calculated relative to the control group.
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Treated KFs were harvested for total RNA extraction using a SteadyPure Universal RNA extraction kit II (Accurate Biology, AG21027) according to the manufacturer’s instructions. Complementary DNA (cDNA) was then synthesized from 500 ng total RNA per sample using an Evo M-MLV RT mix kit (Accurate Biology, AG11728), followed by qPCR using a SYBR Green Pro Taq HS qPCR kit (Accurate Biology, AG11701). The housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was used as an internal control and the qPCR primers used are listed in Table 1.

Primers used in quantitative PCR analysis

GenePrimer sequence (5’–3’)
COL-3Forward: TGGTGTTGGAGCCGCTGCCAReverse: CTCAGCACTAGAATCTGTCC
FNForward: GCCACTGGAGTCTTTACCACAReverse: CCTCGGTGTTGTAAGGTGGA
α-SMAForward: CATCATGCGTCTGGATCTGGReverse: GGACAATCTCACGCTCAGCA
MMP-1Forward: ACAACTGCCACAAGCAATGAGReverse: CTGTCCCTGAACAGCCCAGTACTTA
TIMP-1Forward: AAGACCTACACTGTTGGCTGTGAGReverse: GTCCGTCCACAAGCAATGAG
TGF-β1Forward: AAGGACCTCGGCTGGAAGTGReverse: CCGGGTTATGCTGGTTGTA
TGF-β2Forward: CTGGATGCAGCCTATTGCTTReverse: TGGTGAGCGGCTCTAAATCT
TGF-β3Forward: AACCTGAGCACCTCCAGGACReverse: GCTGCACTTGCAGGATTTG
GAPDHForward: TCACCATCTTCCAGGAGCGReverse: CTGCTTCACCACCTTCTTGA
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The levels of expression for gyrb, hlα, crtM, seA, and seB were analyzed using reverse transcription (RT) followed by real-time quantitative PCR (qPCR). Briefly, a total of 2 mL of S. aureus grown with different concentrations of myricetin (0 to 32 μg/mL) was incubated at 37 °C with shaking at 200 rpm for 16 h. S. aureus grown without myricetin was treated in the same manner and used as a negative control. Subsequently, each sample was centrifuged at 10,000× g for 10 min to collect the bacteria. RNA was extracted from each culture using the SteadyPure RNA Extraction Kit (Accurate Biology, Changsha, Hunan, China), and its purity and concentration were determined using a SpectraMax ABS Puls (Molecular Devices, Shanghai, China). The RNA was then reverse-transcribed using the Evo M-MLV RT Mix Kit (Accurate Biology, Changsha, Hunan, China). The resulting cDNA was quantified using 2 × SYBR Green Abstart qPCR Mix from Sangon Biotech in a qTOWER3 G real-time PCR system (Analytic Jena, Jena, Germany). The primers used are listed in Table 2. The RNA transcript levels were determined using the 2−ΔΔCT method. The experiment was conducted in triplicate for each reaction.
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Total RNA was extracted from lung tissue and cells using the TRIzol reagent (Takara Biotechnology) according to the manufacturer’s instructions, and then reverse transcribed into cDNA using the Evo M-MLV RT Mix Kit (Accurate Biology, AG11728) or miRNA 1st strand cDNA synthesis kit (Accurate Biology, AG11716). Subsequently, qPCR was performed using the SYBR Green Premix Pro Taq HS qPCR Kit II (Accurate Biology, AG11702). The relative expression levels of miRNAs or mRNAs were normalized to U6 small nuclear RNA (snRNA) or β-actin using the 2-ΔΔCt comparison method. Primers used in this study are listed in Additional file 1: Table S3.
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Total RNA was prepared from the serum, AML‐12 cells or mice liver tissues adopting Steadypure Universal RNA Extraction Kit (Accurate Biotechnology) separately, followed by the production of cDNA utilizing Evo M‐MLV RT Mix Kit (Accurate Biotechnology). PCR amplification was performed on the CFX Opus 96 Real‐Time PCR system (IMH‐Bio) via the SYBR® Green Pro Taq HS kit (Accurate Biotechnology). The calculation of relative gene expression was achieved by virtue of the 2−ΔΔCq method. GAPDH expression was used for normalization.
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Total RNA was extracted with the FastPure Cell/Tissue Total RNA Isolation Kit V2 from Vazyme (Nanjing, China) according to the manufacturer’s protocol. Reverse transcription was performed using an Evo M-MLV RT Mix Kit with the gDNA Clean for qPCR AG11728 or miRNA 1st strand cDNA synthesis kit AG11717 (Accurate Biotechnology, Hunan, China). qRT-PCR was performed using an ABI 7500 Fast Real-Time PCR system (Applied Biosystems, MA, USA) with an SYBR Premix Kit AG11701 (for mRNA) or AG11702 (for miRNA) (Accurate Biotechnology, Hunan, China). The levels of gene expression were quantified by the 2−ΔΔCt method. GAPDH was used as the reference gene for mRNA, and U6 was used as the control for miRNA. The primer sequences used are listed in Table 1, and they were purchased from Sangon Biotech (Shanghai, China).
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According to the instructions, total RNAs were extracted from the bilateral dorsal horns using RNAex Pro Reagent (AG21102, Accurate Biotechnology, Hunan, China). Complementary DNA (cDNA) was synthesized using Evo M-MLV RT Mix Kit (AG11728, Accurate Biotechnology, Hunan, China). Polymerase chain reaction (PCR) amplifications were conducted using SYBR® Green Premix Pro Taq HS qPCR kit (AG11701, Accurate Biotechnology, Hunan, China). Real-time fluorescent quantitative PCR was carried out using Light Cycler® 480 II (Roche, Switzerland). β-actin was served as the internal reference for normalization. The mRNA levels of RIPK3, NLRP3, and caspase-1 were calculated using the 2−ΔΔCT method. The primers for NLRP3, RIPK3, caspase-1, and β-actin are shown in Table 1.
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