A total of 20 matched HNSCC and paracancerous tissues were obtained from Wuhan Union Hospital. Pathologists histopathologically confirmed the diagnosis of all tissues. All patients had not received chemotherapy, radiotherapy, targeted drugs, immunotherapy, or Chinese herbal medicine. Patients were not diagnosed with malignancy at other sites or with other serious underlying diseases. The Ethics Committee of Wuhan Union Hospital authorised this research (No: 20220076). All patients signed the informed consent form before surgery. The specimens were removed and rapidly frozen in liquid nitrogen and stored in a low-temperature refrigerator at −80°C for subsequent studies. Total RNA was extracted with a RNeasy mini kit (Axygen, United States) according to the manufacturer’s instructions. cDNA was reverse transcribed by a PrimeScript RT reagent kit with gDNA Eraser (TaKaRa, Japan, Code No. RR047A). The RNA and cDNA of each sample were analyzed by a GeneQuant pro RNA Calculator to assess the concentrations and purity. Quantitative real-time PCR was performed with real-time SYBR Green PCR reagents (Q311-02, Vazyme, Nanjing, China) and the 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA). The abundance of different transcripts was assessed in triplicates.
Rneasy mini kit
Manufactured by Corning
Sourced in United States
The RNeasy Mini Kit is a set of laboratory equipment designed for the isolation and purification of total RNA from a variety of sample types. The kit utilizes a silica-based membrane technology to efficiently capture and purify RNA, allowing for reliable and consistent results.
Automatically generated - may contain errors
Lab products found in correlation
Primescript rt reagent kit with gdna eraser, by Takara Bio (2 mentions)
7300 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Real time sybr green pcr reagents, by Vazyme (1 mentions)
Colchicine, by Beyotime (1 mentions)
Automatic chemical analyzer, by Hitachi (1 mentions)
Light microscopy, by Hitachi (1 mentions)
Viia 7 system, by Thermo Fisher Scientific (1 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Sybr premix ex taq 2, by Takara Bio (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Rnasimple total rna kit, by Tiangen Biotech (1 mentions)
4 protocols using rneasy mini kit
1
Transcriptomic Analysis of HNSCC Tissues
2
Hepatoprotective Effects of CBD
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The following reagents were used: CBD (Sigma, United States); colchicine (Beyotime, China); CCl4 (Aladdin, China); peanut oil (Yuanye, China); ELISA kits (Novus, United States); hematoxylin and eosin (HE) and Masson assay kits (Beyotime, China); aspartate aminotransferase (AST) and hyaluronic acid (HA) kits (Nanjing Jiancheng Bioengineering Institute, China); RIPA lysis buffer and a BCA kit (Solaibao, China); COX-2, p-IκBα, and IκBα antibodies (Wanleibio, China); p-NF-κB, NF-κB, p-p38 MAPK, and p-38 MAPK antibodies (CST, United States); TGF-β1, α-SMA, COL-I, PPAR-α and GAPDH antibodies (Abcam, United States); horseradish peroxidase-labeled secondary antibodies (Bioprimacy, China); chemiluminescence (ECL) color developing solution (Merck, United States); an RNeasy mini kit (Axygen, United States); a PrimeScript RT reagent kit with gDNA Eraser and SYBR Green Master Mix (Takara, Japan); an automatic chemical analyzer (Hitachi, Japan); and light microscopy (Nikon, Japan).
3
Quantitative Real-Time PCR Analysis of Liver Gene Expression
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Total RNA was extracted from frozen liver samples using the RNeasy Mini Kit (Axygen, USA). cDNA was synthesized from 1 μg of total RNA using the PrimeScript RT reagent kit with gDNA Eraser (Takara, Japan). Quantitative real-time PCR was carried out on a ViiA 7 system (Applied Biosystems, USA) in 20 μl reactions containing 2 μl of cDNA (1:10 dilution), 400 nM of primers (All primers were obtained from Sangon, China) and PowerUp SYBR Green Master Mix (Applied Biosystems, USA) according to the manufacturer’s instructions. Data are expressed as the ratio of the target gene level to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the treated group relative to the sham group and were quantified using the comparative cycle threshold Ct method (2-▵▵CT). The gene primer sequences were as follows:
TNF-α-- 5’- GCGACGTGGAACTGGCAGAAG-3’,
5’-GCCACAAGCAGGAATGAGAAGAGG-3’
IL-6– 5’- ACTTCCATCCAGTTGCCTTCTTGG-3’,
5’- TTAAGCCTCCGACTTGTGAAGTGG-3’
IFN-γ– 5’- CAGGCCATCAGCAACAACATAAGC-3’,
5’- AGCTGGTGGACCACTCGGATG-3’
GAPDH– 5’-AGGTCGGTGTGAACGGATTTG-3’,
5’-TGTAGACCATGTAGTTGAGGTCA-3’.
4
RNA Isolation and qRT-PCR Analysis
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Total RNA was isolated from cells and tissues using RNeasy Mini Kit (Axygen,NY,USA) and RNA simple Total RNA Kit (Tiangen, China) according to standard protocol. cDNA was generated using the PrimeScript RT reagent Kit (Takara, Japan). qRT-PCR was conducted with SYBR Premix Ex Taq™ II (Takara, Japan) on StepOne plus Real-Time PCR system. The primers for all genes were listed below:
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