Blood from the patients was collected in an ethylene diamine tetraacetic acid (EDTA)-coated bottle and stored at −80°C, while total RNA was extracted by chloroform-isopropanol extraction. Specifically, the samples were subjected to vortex at room temperature for 15 min followed by centrifugation at 10,000 × g and 4°C for 10 min. The supernatant was then mixed with 200 μL chloroform for 15 min and centrifuged at 12,000 × g and 4°C for 15 min. The supernatant was mixed with 500 μL isopropanol for 10 min, and the mixture was centrifuged at 12,000 × g and 4°C for 10 min. Being washed twice with 75% ethanol (1 mL), the mixture was centrifuged again at 7,500 × g for 5 min. The precipitate was dissolved again in 30 mL RNAse-free water. The purity and concentration of the total RNA were determined using a dual-beam ultraviolet spectrophotometer (Eppendorf, Germany).
Dual beam ultraviolet spectrophotometer
Manufactured by Eppendorf
Sourced in Germany
The Dual-beam Ultraviolet Spectrophotometer is a laboratory instrument designed to measure the absorption of ultraviolet light by a sample. It utilizes two separate light beams, one passing through the sample and the other through a reference, to quantify the amount of light absorbed by the sample.
Automatically generated - may contain errors
Lab products found in correlation
Sybr premix real time pcr reagent, by Takara Bio (1 mentions)
Abi 7900 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Onestep primescript mirna cdna synthesis kit, by Qiagen (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (1 mentions)
3 protocols using dual beam ultraviolet spectrophotometer
1
Total RNA Extraction from Whole Blood
2
Quantification of miR-107 and BDNF expression
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Total RNA of the cultured cells and the tissues was extracted using TRIzol (Invitrogen) according to the manufacturer's instructions. The purity and concentration of total RNA were determined by a dual-beam ultraviolet spectrophotometer (Eppendorf, Hamburg, Germany). Then, a total of 3 μg of mRNA was reverse transcribed to single-stranded cDNAs using a PrimeScript® RT reagent kit (Takara Biotechnology Co., Ltd., Dalin, China). qRT-PCR for miR-107 and BNDF were performed using SYBR premix real-time PCR Reagent (Takara) under an ABI7900 real-time PCR system (Applied Biosystems, Foster City, CA, USA). The primers for miR-107 and U6 were brought from Applied Biosystems. The primers for BDNF and β-actin used in this study were as described previously (20 (link)). U6 RNA was used to normalize the miR-107 RNA levels, and β-actin was used to normalize the level of BDNF mRNAs. The comparative 2−ΔΔCt method was employed for relative quantification.
3
Quantifying miR-338-3p in Ovarian Cancer Cells
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Total RNA was extracted from fresh tissues sample and cells (HOSEpiC, SKOV3, OVCAR3 and A2780) using the mirVana miRNA Isolation kit (Ambion, USA), according to the manufacturer's instructions. The purity and concentration of RNA were determined using a dual-beam ultraviolet spectrophotometer (Eppendorf, Hamburg, Germany). Then, the RNA was reversely transcribed into cDNA using One Step Prime script miRNA cDNA Synthesis kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. Then miR-338-3p was quantified as described by Chen et al (18) . U6 snRNA was used as an endogenous control. The comparative 2 -∆∆Ct method was used for relative quantification and statistical analysis.
Transfection of cells with miR-338-3p. The miR-338-3p mimic or corresponding negative control (miR-NC) were purchased from Shanghai GenePharma (Shanghai, China). To transfect cells, 50 nM of miR-338-3p or miR-NC was diluted in 500 µl of serum-free media and 5 µl of Lipofectamine 2000 reagent (Invitrogen, Grand Island, NY, USA) according to the manufacturer's instructions. The transfection mixture was added to 1x10 6 cells in a 60-mm dish containing 4 ml medium supplemented with 10% FbS. The cells were harvested 24, 48 and 72 h after transfection and prepared for the subsequent study. Transfection efficiencies were evaluated in every experiment by qRT-PCR 48-h post-transfection.
Transfection of cells with miR-338-3p. The miR-338-3p mimic or corresponding negative control (miR-NC) were purchased from Shanghai GenePharma (Shanghai, China). To transfect cells, 50 nM of miR-338-3p or miR-NC was diluted in 500 µl of serum-free media and 5 µl of Lipofectamine 2000 reagent (Invitrogen, Grand Island, NY, USA) according to the manufacturer's instructions. The transfection mixture was added to 1x10 6 cells in a 60-mm dish containing 4 ml medium supplemented with 10% FbS. The cells were harvested 24, 48 and 72 h after transfection and prepared for the subsequent study. Transfection efficiencies were evaluated in every experiment by qRT-PCR 48-h post-transfection.
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