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1st strand cdna synthesis kit

Manufactured by Vazyme
Sourced in China

The 1st Strand cDNA Synthesis Kit is a laboratory tool used for the reverse transcription of RNA to complementary DNA (cDNA). This kit provides the necessary reagents and enzymes to convert RNA into single-stranded cDNA, which can then be used for various downstream applications such as gene expression analysis, cloning, and sequencing.

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9 protocols using 1st strand cdna synthesis kit

1

Evaluating mRNA Expression Profiles in ccRCC

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To examine the expression level of the identified FPTOSs in ccRCC sample, we further carried out RT-PCR experiments to compare the mRNA expression difference between human ccRCC tumor specimen and adjacent normal specimen. Moreover, the mRNA expression of FPTOSs in human normal renal proximal tubular cell line (HK2), human renal clear cell carcinoma cell lines (786-O, OS-RC-2) were also evaluated. Cells was purchased from Shanghai Cell Bank Type Culture Collection Committee (Shanghai, China) and incubated in RPMI-1640 medium containing 10% fetal bovine serum (FBS). The total RNA was extracted using Trizol reagent and then transcribed into cDNA using 1st Strand cDNA Synthesis Kit (Vazyme, China). RT-PCR method was performed via qPCR SYBR Green Master Mix (Vazyme, China) in a QuantStudio™ 6 Flex Real-Time PCR System. The result was normalized to housekeeping gene GAPDH, and the selected primers for the FPTOSs were listed in Table S1.
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2

Reverse Transcription qPCR Protocol

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RT‐qPCR was performed as described in our previous study.52 Briefly, total RNA was extracted using RNAiso Plus (Takara Bio). A total of 1 μg RNA was reverse transcribed into complementary DNA (cDNA) by a 1st Strand cDNA Synthesis kit (Vazyme). RT‐qPCR assay was performed using a SYBR qPCR Master Mix (Vazyme) in a CFX96 Real‐Time System (Bio‐Rad Laboratories). Mouse or human β‐actin was used as an internal control. The sequences of the primers used are summarized in Table 1.
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3

RT-qPCR Analysis of Fungal Transcripts

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Total RNA was isolated from mycelia grown on CM plates with cellophane overlays and used for both RT‐qPCR assays and RNA‐Seq analysis. For RT‐qPCR assays, 1st Strand cDNA Synthesis Kit (Vazyme) was used to generate complementary DNA (cDNA). The qPCR assay was performed on the CFX Connect Real‐time PCR System (Bio‐Rad) using TransStart Tip Green qPCR SuperMix (TransGen Biotech) (Li et al., 2011 (link)). The β‐tubulin gene (MGG_00604) was used as the internal control, and the expression level of target genes was calculated using the 2−∆∆Ct method (Livak & Schmittgen, 2001 (link)). The RT‐qPCR assay was repeated three times with independent samples. Data were analysed with GraphPad software. The primers used are listed in Table  S1.
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4

Validation of RNA-Seq Transcriptome Profiling

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Initially, five upregulated and eight downregulated unigenes were selected to support the accuracy of the RNA-seq results. Total RNA of non-overwintering and overwintering samples were extracted using the methods described above. cDNA was synthesized using 1 μg total RNA template following the instructions outlined by the 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China). The expression levels of 13 genes were determined by qPCR using the Hieff UNICON qPCR SYBR Green Master in the Applied Biosystems 7500 System (USA) according to the manufacturer’s instructions. The PCR procedure was as follows: 5 min at 95°C, 40 cycles of 10 s at 95°C, and 40 s at 60°C, followed by melting curve analysis. Sequence and efficiency of the primer is provided in Supp Table S1 (online only). Relative expression levels were measured based on the 2−ΔΔCt method with Ribosomal Protein 10 (RPL10) as the internal control for the normalization of data (Li et al. 2018 ). To verify the reliability of RNA-Seq data, Pearson’s correlation between fold changes (Log2 transformed) in RT-qPCR and RNA-Seq results was analyzed.
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5

Quantitative Analysis of miRNA and mRNA Expression in DVT Patients

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Total RNA from PBMCs of DVT patients and healthy controls or the experimental cell lines was extracted using the TRIzol Reagent (Invitrogen, Carlsbad, United States). miRNAs and mRNAs were reverse transcribed using the 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China) and PrimeScript RT reagent Kit (Toyobo, Osaka, Japan), respectively, according to the manufacturer’s instructions. The relative expression of each RNA was quantified using SYBR Green (Invitrogen, Carlsbad, United States) based qRT-PCR assay on an Applied Biosystems 7500 instrument (Applied Biosystems, Foster, United States). U6 and GAPDH were used as internal controls for miRNA and mRNA expressional normalization, and relative RNA quantification was calculated via the comparative 2−ΔΔCt method. For mRNA and miRNA expressional analysis, the PCR primer sequences used are shown in Supplementary Tables S2, S3. Unless otherwise stated, the amplification reaction for each sample was conducted in triplicate.
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6

Quantification of HOTAIR and miR-130a-3p

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Total RNA samples were extracted from hippocampal tissues using an RNA easy fast tissue/cell kit (TIANGEN, Beijing, China). The purity and concentration were detected by the DanoDrop 2000. The first line of cDNA of HOTAIR was synthesized through 1st strand cDNA synthesis superMix (Yeason, Shanghai, China). For miR-130a-3p, 1st strand cDNA synthesis kit (Vazyme, Nanjing, Jiangsu, China) was adopted to synthesize cDNA. The qRT-PCR assay was performed using the Toyobo SYBR qPCR mix (Toyobo, Osaka, Japan). With GAPDH or U6 as the internal references, the relative content of HOTAIR or miR-130a-3p was calculated by the 2−ΔΔ Ct method.
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7

RNA Extraction and qPCR Analysis

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Total RNAs in cells and tissues were extracted using Trizol reagent (Invitrogen, USA), according to the procedures as previously described [13 (link)]. Subsequently, 1 μg of total RNA was used for reverse-transcription using 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China). Q-PCR was performed using SYBR Green Master Mix (Vazyme, Nanjing, China) on a 7500 Real Time PCR System (Applied Biosystems, CA, USA). The relative gene expression was calculated by the 2-ΔΔCt method. GAPDH or U6 was used as internal references. The primers used in this study were purchased from Shanghai Shenggong Biological Engineering Co., Ltd. and as follows: circNMD3-forward, 5’-GTTTAATGGAGCTTGAGGGT-3’; reverse 5’-GGTCCATGCACATAAGGAAT-3’. NMD3-forward, 5’-AAGTCTCGATTTCGTTCTGCAA-3’; reverse, 5’-CCTTACTCAGAGGGGCTTTGAT-3’. GAPDH-Forward, 5’-GGAGCGAGATCCCTCCAAAAT-3’; reverse, 5’-GGCTGTTGTCATACTTCTCATGG-3’. miR-498-forward, 5’-TTTCAAGCCAGGGGGCGTTTTTC-3’; reverse, 5’-GCTTCAAGCTCTGGAGGTGCTTTTC-3’. U6-forward, 5’-AACGCTTCACGAATTTGCGT-3’; and reverse, 5’-CTCGCTTCGGCAGCACA-3’.
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8

Transcriptomic Analysis of M. oryzae

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RNA samples were isolated from mycelia of M. oryzae strains cultured in liquid MM supplemented with or without propranolol at 28°C for 3 days. The FastPure Plant Total RNA Isolation Kit (Vazyme, China) was used to extract total RNA from samples. RNA samples were sequenced on the BGISEQ-500 sequencer (BGI, China). Data were quality-controlled and mapped to the M. oryzae genome (http://fungi.ensembl.org/index.html). Gene expression and differential expression analyses were performed using featureCounts and DESeq2, respectively. The criteria for DEGs were p ≤ 0.05 and log2FC ≥ 1 (FC, fold change).
For qRT–PCR assays, we used the 1st Strand cDNA Synthesis Kit (Vazyme, China) to generate complementary DNA (cDNA). TransStart Tip Green qPCR SuperMix (TransGen Biotech, China) was used for qRT–PCR assays on the CFX Connect Real-time PCR System (Bio-Rad, USA). The β-tubulin gene (MGG_00604) was used as the internal control to calculate gene expression by the 2−ΔΔCt method. Each treatment was repeated at least three times with independent samples. The primers used are listed in Supplemental Table 1.
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9

Cloning and Phylogenetic Analysis of NtSOS2 from Nitraria tangutorum

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Total ribonucleic acid was isolated from Nitraria tangutorum leaves with an RNA extraction kit (Bioteke, China) and treated with DNase I (Takara, Japan) to remove contaminating desoxyribonucleic acid. cDNA was synthesized with 1st strand cDNA synthesis Kit (Vazyme, China). The NtSOS2 was amplified from Nitraria tangutorum’s cDNA using specific primers which designed by Oligo 7.60 (Cascade, CO 80809, United States) that were listed in Supplementary Table 1. Homologous proteins were searched for using the NCBI blastp program and multiple alignments were performed using DNAMAN v9.0 (Lynnon Corporation, San Ramon, CA, United States) with the deduced amino acid sequences. A phylogenetic tree was constructed through the Neighbor-Joining method, and the bootstrap value was calculated according to 1,000 repetitions using MEGA-X v10.1 (Temple, Philadelphia, PA, United States).
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