RAW264.7 macrophages (1 × 106 cells/well) were cultured in 6-well plates with 24 h incubation, CE with various concentrations (0–100 μg/mL) were added in each well (except control group) for 2 h of pretreatment, and 1 μg/mL LPS was subsequently added for 6 h incubation. Then, total RNA was extracted by using the TransZol Up RNA extraction kit (Transgen Biotech, Beijing, China). The purities and concentrations of RNA were measured using a UV spectrophotometer (Biochrom, UK) with the absorbance 260/280 nm. PrimeScriptTM RT reagent kit with gDNA eraser (Takara, Dalian, China) was used to remove genomic DNA and reverse transcribe 1 μg total RNA to cDNA. The target genes expressions were observed intuitively by the semiquantitative RT-PCR method. Agarose gel stained by GelStain (1%, Transgen Biotech, Beijing, China) was used to analyze the PCR products. In addition, quantitative PCR was performed in 20 μL reaction volumes by qTOWER3G (Analytik Jena, Germany) to further quantify the expression levels of mRNA. The cycling procedure was 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. The expressions of relative genes were analyzed according to the 2−ΔΔCt method. The primer sequences are given in Table 1 , and GAPDH was considered as an internal reference.
Transzol up rna extraction kit
Manufactured by Transgene
Sourced in China
The TransZol Up RNA extraction kit is a laboratory product designed for the efficient extraction and purification of high-quality RNA from a variety of biological samples. It utilizes a proprietary method to effectively isolate RNA while removing contaminants.
Automatically generated - may contain errors
Lab products found in correlation
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Gelstain, by Transgene (1 mentions)
Uv spectrophotometer, by Harvard Bioscience (1 mentions)
Primescripttm rt reagent kit with gdna eraser, by Takara Bio (1 mentions)
Qtower3g, by Analytik Jena (1 mentions)
Qubit rna hs assay kit, by Thermo Fisher Scientific (1 mentions)
Iq sybr green supermix, by Bio-Rad (1 mentions)
Transtart tip green qpcr supermix, by Transgene (1 mentions)
Peasy blunt zero, by Transgene (1 mentions)
All in one first strand cdna synthesis supermix for pcr, by Transgene (1 mentions)
4 protocols using transzol up rna extraction kit
1
Macrophage Inflammatory Response Modulation
2
RNA Extraction, cDNA Synthesis, and RT-PCR for Stress Gene Expression
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RNA extraction and determination of RNA concentration (ng/ µL), cDNA synthesis and RT-PCR reaction were implemented according to the manufacturer's instructions for the kits mentioned hereinafter. RNA extraction from cheese was performed with the TransZol Up RNA extraction kit (TransGen Biotech, China). The amount of RNA was determined with the Qubit RNA HS Assay Kit (Thermo Fisher Scientific, USA). cDNA was synthesized from two separate RNA extractions concentration adjusted to 30 ng/µL. A high-capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, USA) was used for cDNA synthesis. For each reaction, adjusted concentration of 10 μL RNA was added to the mixture containing 10X RT Buffer (2.0 μL), 25X dNTP Mix (100 mM, 0.8 μL), 10X RT Random Primers (2.0 μL), MultiScribeTM Reverse Transcriptase (1 μL), Nuclease-free H2O (4.2 μL). Reverse Transcriptase PCR reaction mixture containing 3.5 µL iQ™ SYBR® Green Supermix (BioRad, USA), 0.5 µL cDNA template, 0.5 µL forward primer (10 µM), 0.5 µL reverse primer (10 µM), and 5 µL sterile water the mixture with a volume of 10 µL was prepared. Each analysis was run with a DNA-free control. Livak and Schmittgen's 2 -ΔΔCT calculation method was used to determine stress-related gene expression level changes [26] (link). Primer sequences are given in Table 1.
3
Argonaute Protein Expression Analysis
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The TransZol UP RNA extraction kit and TranStart Tip Green qPCR SuperMix were acquired from TransGen Biotech (Beijing, China), and diethyl pyrocarbonate and DEPC were purchased from Beyotime (Beijing, China). Passive Reference Dye and RevertAid First Strand cDNA Synthesis Kit were procured from Thermo Fisher Scientific. Argonaute2 (Bs-20459R) and Argonaute3 (Bs-12517R) antibodies were purchased from Bioss (Beijing, China) and the immunohistochemical kit (sp-002) and DAB (3,3N-diaminobenzidine) chromogenic reagent kit were acquired from Solarbio Life Sciences (Beijing, China). All reagents and kits were used as received without modification.
4
Cloning and Sequencing of ApCtf1β Genes
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According to the manufacturer’s instructions, the total RNA of A. phaeospermum was extracted using the TransZol Up RNA Extraction Kit (TransGen). The extracted products were tested for purity and integrity via agarose electrophoresis and microspectrophotometry. According to the manufacturer’s instructions, RNA that met the target requirements was reverse-transcribed to synthesize cDNA for backup using the All-in-One First-Strand cDNA Synthesis Super Mix for PCR (TransGen a). The homologous primers ApCtf1β-F/ApCtf1β-R (Supplementary Table S1 ) were designed based on the ApCtf1β1 and ApCtf1β2 genes (MK789640 and MK789641) in the NCBI database. PCR amplification was performed using A. phaeospermum cDNA as the template. The PCR products were detected via 1% agarose gel electrophoresis, recovered, and ligated to the cloning vector pEASY-Blunt Zero (TransGen) to obtain the recombinant vector. The ligated products were transformed into receptor cells DH5α. Positive transformants were picked following verification via colony PCR and sent to Tsingke for sequencing to confirm that the target genes were not mutated.
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