The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). Surgical specimens from 5 LUSC patients were collected from the Specimen Bank of Hangzhou First People’s Hospital with approval by the Institutional Review Board of Hangzhou First People’s Hospital {[2015](043)-01}. Informed consent was waived since this study was observational and presents no more than minimal risk of harm to subjects and involves no procedures for which written consent is normally required outside the research context. Total RNA was extracted from 30 mg tissue with the AxyPrep Multisource RNA Miniprep Kit according to the manufacturer’s protocol (Axygen, Tewksbury, MA, USA). cDNA from 1 µg RNA was prepared using the PrimeScriptTM RT Reagent Kit (Takara, Japan). Quantitative real-time PCR (RT-qPCR) was performed using TB Green Premix Ex Taq (Takara, Japan) and a 7500 System (Applied Biosystems, Singapore). All RT-qPCR assays were carried out in 3 biological replicates, and the average cycle threshold (CT) was used. Target genes were normalized using the RPLP0 gene, and the relative expression of the target genes was calculated as follows: 2−ΔCT, where ΔCT = Avg. CT (Target gene) − Avg. CT (RPLP0). The primers used for quantitative real-time PCR are listed in Table 1 .
Axyprep multisource rna miniprep kit
Manufactured by Corning
Sourced in United States, China
The AxyPrep Multisource RNA Miniprep Kit is a laboratory equipment product designed for the purification of total RNA from various sample types, including animal cells, tissues, bacteria, and yeast. The kit utilizes a silica-based membrane technology to efficiently capture and purify RNA, ensuring high-quality RNA suitable for downstream applications such as RT-PCR, Northern blotting, and RNA sequencing.
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Lab products found in correlation
Tb green premix ex taq, by Takara Bio (3 mentions)
Fastquant rt kit with gdnase, by Takara Bio (3 mentions)
7500 system, by Thermo Fisher Scientific (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Cfx96 real time pcr detection system, by Roche (2 mentions)
Kapa sybr fast qpcr kit, by Roche (2 mentions)
Transscript one step gdna removal and cdna synthesis supermix, by Transgene (2 mentions)
Primescripttm rt reagent kit, by Takara Bio (1 mentions)
Taq pcr master mix, by Vazyme (1 mentions)
Hiscript 2 1st strand cdna synthesis kit, by Vazyme (1 mentions)
Tip green qpcr supermix, by Transgene (1 mentions)
Novaseq 4000, by Illumina (1 mentions)
Il 10, by Sangon (1 mentions)
Sybr green kit, by Transgene (1 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Sirt1, by Sangon (1 mentions)
Transscript first strand cdna synthesis supermix, by Transgene (1 mentions)
β actin, by Sangon (1 mentions)
Tnf α, by Sangon (1 mentions)
19 protocols using axyprep multisource rna miniprep kit
1
LUSC Tissue RNA Extraction and qPCR Analysis
2
Viral Detection in Chicken Liver Tissues
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The total RNA was extracted from the liver tissue sample suspension using the AxyPrep Multisource RNA Miniprep Kit (Axygen, USA) and cDNA was synthesized using the HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, China). For detection of ARV, the PCR was performed with specific primers of S2 gene listed in Table 1 (26 (link)). The PCR reaction volume was 25 μl containing 12.5 μl of 2× Taq PCR Master Mix (Vazyme, China), 1 μl of each primer, 9.5 μl of double-distilled water (ddH2O), and 1 μl of the cDNA template. The PCR cycling conditions for the S2 gene amplifications were as follows: 1 cycle of 94°C for 5 min, 35 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for 1 min, followed by a final extension step of 72°C for 10 min. In addition, PCR detection for chicken infectious anemia virus (CIAV), reticuloendotheliosis virus (REV), A subgroup of avian leukosis virus (ALV-A), B subgroup of avian leukosis virus (ALV-B), J subgroup of avian leukosis virus (ALV-J), and K subgroup of avian leukosis virus (ALV-K) was performed. The primers used are listed in Table 2 .
3
RNA Extraction and RT-qPCR Analysis of U87 and GSCs
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RNA was extracted from cells (both U87 and GSCs) using the AxyPrep Multisource RNA Miniprep Kit (Axygen; Corning, Inc.). cDNA was performed with the GoScript™ Reverse Transcriptase System kit (cat. no. A5001, Promega Corporation), according to the manufacturer's instructions-PCR amplification was performed using Tip Green qPCR SuperMix (TransGen Biotech Co., Ltd.) in a 20 µl reaction containing Tip Green qPCR SuperMix buffer, primers and diluted cDNA. The primer sequences are listed in Table I . The PCR cycling conditions were as follows: 94˚C for 30 min, followed by 40 cycles of 94˚C for 30 sec, 50-60˚C for 30 sec and 72˚C for 1 min/kb. All relative expression values were normalized to GAPDH levels using the 2-∆∆Cq method (36 (link)).
4
Quantification of Fungal Gene Expression
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The WT strain and OE-275 mutant were cultured in PDA medium at 28 °C for 6 days. The vegetative hyphae were harvested in 9-cm Petri dishes, which were then incubated at 28 °C for 3 and 5 d. After induction, hyphae were collected and frozen immediately in liquid nitrogen. Total RNA was extracted from all samples with the AxyPrep Multisource RNA Miniprep Kit (Axygen, Jiangsu, China). The extracted RNA was then reverse transcribed to cDNA with the FastQuant RT Kit with gDNase (Takara, Kusatsu, Japan). The cDNA was used as the template for a qRT-PCR assay, which was conducted to analyze the transcription levels of candidate genes. The gene-specific qRT-PCR primers were designed with Primer3 software, and the β-tubulin gene (Tub, AOL_s00076g640) was used as an internal standard. The qRT-PCR analysis was performed as previously described [16 (link)]. The relative transcription level (RTL) of each gene was calculated as the ratio of the transcription level in the deletion mutant to the transcription level in the WT strain at a given timepoint according to the 2−ΔΔCt method [16 (link)].
5
Transcriptome analysis of A. oligospora response to nematodes
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WT and Δfus3 strains were grown on cellophane-overlaid PDA plates at 28°C for 5 days. Approximately 600 nematodes were then added to each plate for 0, 12, 24, and 36 h of induction, and three replicates were used for each sample. After collecting the samples, RNA was extracted with the AxyPrep multisource RNA miniprep Kit (Axygen, Jiangsu, China), and then sent to Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China) for transcriptome sequencing. The final cDNA library was sequenced on an Illumina Novaseq 4000 platform. The clean reads were mapped to the A. oligospora (ATCC 24927) genome sequence. The gene expression changes were evaluated and the DEGs were identified with an FDR value of ≤0.05. High-throughput sequencing data were analyzed using the OmicShare online platform (www.majorbio.com ).
6
Comprehensive Gene Expression Analysis
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Total RNA was extracted with an Axyprep multisource RNA miniprep kit (Axygen, America), and cDNA was synthesized using TransScript First-Strand cDNA Synthesis SuperMix (TransGen Biotech, Beijing). Quantitative real-time PCR was performed using a SYBR Green kit (TransGen Biotech, Beijing) with a StepOnePlus real-time PCR system (ABI), and the primer sets used for MTTP, ApoA1, ApoB, ApoC2, CD31, TGFβ, TSP1, VEGFR1, IL-1β, TNFα, IL-6, IL-10, CCL2, PPARα, SIRT1, and β-actin (Sangon Biotech, Shanghai) are listed in Table 1 . Relative gene expression was measured with triplicates for each sample and normalized to β-actin.
7
Quantifying TLR7, TLR8, and MyD88 Expression
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To measure the relative expression of TLR7, TLR8, and MyD88, RNA was extracted from 5 × 105 cells using AxyPrep Multisource RNA Miniprep Kit (Axygen) and measured by Nano-drop. An equal amount of RNA was reverse-transcribed into cDNA through the iScript cDNA Synthesis Kit (Bio-red). Samples were quantified by PCR with SYBR Green (Applied Biosystem). All specific primers used for the analysis were designed by qPrimerDB (Lu et al., 2018 (link)), and the sequences were shown as follows:
To measure the HIV viral load, viral RNA from supernatants was extracted by MagaBio Plus Viral DNA / RNA Purification Kit (BIOER). The reverse-transcription and analysis of RNA copies were performed using the HIV-1 Real-time PCR detection kit (BIOER).
To measure the HIV viral load, viral RNA from supernatants was extracted by MagaBio Plus Viral DNA / RNA Purification Kit (BIOER). The reverse-transcription and analysis of RNA copies were performed using the HIV-1 Real-time PCR detection kit (BIOER).
8
Transcriptomic Analysis of Fungal Mutants
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WT and mutant strains were incubated in PD (200 g potatoes and 20 g glucose per 1 L) broth medium at 28°C for 5 and 7 days, respectively. Three replicates were used for each sample. Mycelial samples were collected and frozen in liquid nitrogen, followed by sequencing by Majorbio Bio-pharm Technology Co. Ltd (Shanghai, China). Then, the Majorbio Cloud platform was used to evaluate the data (www.majorbio.com ). RNA was extracted using the AxyPrep Multisource RNA miniprep kit (Axygen, Jiangsu, China), transcriptome results were verified by RT-qPCR, and the selected genes and relative primers are listed in Table S3 (23 (link), 24 (link)). Finally, differentially expressed genes (DEGs) were screened out using P ≤ 0.05 and Log2 (fold change) ≥ 2 as thresholds. The GO and KEGG databases were used to functionally assess the selected DEGs (56 (link)).
9
Quantification of Sporulation Genes in Mutant Strains
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To analyze the expression of sporulation-related genes in WT and mutant strains, total RNAs were extracted from 3-, 5-, and 7-day cultures grown on TYGA by using an AxyPrep multisource RNA miniprep kit (Axygen, Jiangsu, China), and then reverse-transcribed into cDNAs by using a FastQuant RT kit with gDNase (TaKaRa). The cDNA samples of each strain were used as the template to assess the transcript level of each gene by using SYBRH Premix Ex Taq (TaKaRa) and performing RT-PCR with paired primers (Supplementary Table S2 ); β-tubulin served as an internal standard, and the 2–ΔΔCt method (Livak and Schmittgen, 2001 (link)) was used for quantification. The relative transcription level (RTL) of each gene was calculated as the ratio of the transcription level in the deletion mutant to the transcription level in the WT strain at a given time point.
10
Quantifying TNFR Superfamily mRNA Levels
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The total RNA was extracted from BV2 cells using Axygen® AxyPrep™ Multisource RNA Miniprep Kit following the manufacturer’s instruction, and the RNA was reverse-transcribed into cDNA using First Strand cDNA Synthesis Kit. Standardization was performed with GAPDH as the endogenous control. The real-time PCR was performed with the SYBR green two-step qRT-PCR kit. Real-time PCR was analyzed with the TAKARA Thermal Cycler Dice™ Real time system (TAKARA, Dalian, China). mRNA levels were determined using the 2−δδCt method. Results are expressed as the fold changes of mRNA in untreated cells (CTL).
The specific primer were as follows: for TACI forward (F) 5-GTGTGGCCACTTCTGTGAGA-3 and reverse (R) 5-CTGGTGCCTTCCTGAGTTGT-3; BAFFR forward (F) 5-GTGCCTTCAGATGGTTGGAT-3 and reverse (R) 5-CCATACCTCCAGCCCAGTAA-3; BCMA forward (F) 5-ATCTTCTTGGGGCTGACCTT-3 and reverse (R) 5-CTTTGAGGCTGGTCCTTCAG-3; GAPDH forward (F) 5-CATGGCCTTCCGCGTTCCTA-3 and reverse (R) 5-ATGCCTGCTTCACCACCTTCT-3.
The specific primer were as follows: for TACI forward (F) 5-GTGTGGCCACTTCTGTGAGA-3 and reverse (R) 5-CTGGTGCCTTCCTGAGTTGT-3; BAFFR forward (F) 5-GTGCCTTCAGATGGTTGGAT-3 and reverse (R) 5-CCATACCTCCAGCCCAGTAA-3; BCMA forward (F) 5-ATCTTCTTGGGGCTGACCTT-3 and reverse (R) 5-CTTTGAGGCTGGTCCTTCAG-3; GAPDH forward (F) 5-CATGGCCTTCCGCGTTCCTA-3 and reverse (R) 5-ATGCCTGCTTCACCACCTTCT-3.
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