Polyetheretherketone (PEEK) sheets were provided by Jiangsu Junhua High Performance Specialty Engineering Plastics (PEEK) Products Co., Ltd (Changzhou, China). The PEEK substrate was machined either into a disc format (Φ15 mm × 1 mm) for surface characterization and in vitro studies, or a plate format (6 mm × 2.8 mm × 1 mm) for in vivo animal experiments. Prior to use, all the samples were washed sequentially with 2-propanol, acetone, ethanol and ultrapure water in an ultrasonic cleaner, and finally dried under vacuum overnight. Vinylphosphonic acid (VPA) was provided by TCI (Shanghai) Development Co., Ltd. α-MEM, fetal bovine serum, and penicillin/streptomycin were purchased from Hyclone. Cell Counting Kit-8 (CCK-8) was provided by Dojindo (Kumamoto, Japan). BCA kit and ALP assay kit were purchased from Nanjing Jiancheng Bioengineering Institute. Alizarin Red, cetylpyridinium chloride, and Sirius Red were obtained from Sigma. TRIZOL reagent was from Invitrogen Life Technologies. PrimeScript RT reagent kit with gDNA Eraser and TB Green premix EX Taq II PCR kit were obtained from Takara (Dalian, China).
Tb green premix ex taq 2 pcr kit
Manufactured by Takara Bio
Sourced in China
The TB Green Premix EX Taq II PCR kit is a reagent kit designed for PCR amplification. It contains a DNA polymerase, buffer, and a fluorescent dye that binds to double-stranded DNA to enable real-time detection of amplification.
Automatically generated - may contain errors
Lab products found in correlation
Primescript rt reagent kit, by Takara Bio (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Cetylpyridinium chloride, by Merck Group (1 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (1 mentions)
Alizarin red, by Merck Group (1 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (1 mentions)
Sirius red, by Merck Group (1 mentions)
Alp assay kit, by Nanjing Jiancheng (1 mentions)
Bca kit, by Nanjing Jiancheng (1 mentions)
Cfx connect real time pcr detection system, by Bio-Rad (1 mentions)
Rnaiso plus, by Takara Bio (1 mentions)
Cfx96 real time pcr system, by Bio-Rad (1 mentions)
Nanodrop 2000 microspectrophotometer, by Thermo Fisher Scientific (1 mentions)
5 protocols using tb green premix ex taq 2 pcr kit
1
PEEK Surface Functionalization and Characterization
2
RT-qPCR Analysis of Gene Expression
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The primers used in the study are listed in Table 2 . All the primers were designed and synthesized by Tsingke Biological Technology (Beijing, China). Quantitative real-time polymerase chain reaction (RT-qPCR) was carried out using the CFX Connect Real-Time PCR Detection System (Bio-Rad, Shanghai, China), in which the amplification and detection steps were combined. The reactions were performed using the TB Green Premix Ex Taq II PCR kit (TaKaRa; Dalian, China). All the assays were performed using three biological replicates. A single qPCR reaction was performed in a 20 µL volume containing 10 µL SYBR Green Master Mix, 0.8 µL of each primer, 2 µL of cDNA sample, and 6.4 µL water free of RNase and DNase.
3
Quantitative Analysis of Gene Expression
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A549 cells (1×105 per well) were seeded in the 6-well plate for total RNA extraction. Total RNA was isolated from clinical samples and cultured cells using RNAiso Plus (Takara Bio, Inc.) according to the manufacturer's instructions. RNA was reverse transcribed using the PrimeScript RT Reagent kit with genomic DNA (gDNA) Eraser (Takara Bio, Inc.). The gDNA elimination reaction was performed for 2 min at 42°C. Reverse transcription was conducted for 15 min at 37°C and 5 sec at 85°C. The qPCR analysis was performed using the CFX96 real-time PCR system (Bio-Rad Laboratories, Inc.) and the TB Green Premix EX Taq II PCR kit (Takara Bio, Inc.). Amplification consisted of an initial 30 sec incubation at 95°C, followed by 40 cycles of 5 sec at 95°C and 30 sec at 60°C. Data were expressed as the relative differences between the control and treated cells after normalization to GAPDH expression. To determine the relative expression levels of miRNAs, the U6 small nuclear RNA was used for normalization. The relative expression levels of the mRNA and miRNA was calculated using the 2−ΔΔCq method (12 (link)). Primers for miR-320a-3p and U6 were purchased from GenePharma Co. Ltd., while those for GAPDH, ACTA2, vimentin, CDH1 were purchased from Takara Bio, Inc. The sequence of the primers used are listed in Table II . All experiments were repeated three times.
4
Quantification of miR-423-5p and mRNA Levels
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Total RNA (1 µg) from airway tissues or BEAS-2B cells (1×106) was used to synthesize complementary DNA (cDNA) with the PrimeScript RT Reagent kit (Takara Biotechnology Co., Ltd.). Subsequently, cDNA was amplified via the TB Green Premix EX Taq II PCR kit (Takara Biotechnology Co., Ltd.). Amplification cycle included an initial 30 sec incubation at 95°C for denaturation, followed by 40 cycles of 5 sec at 95°C for annealing and 30 sec at 60°C for elongation. The small nuclear RNA U6 was utilized for the internal normalization of miR-423-5p levels and GAPDH was utilized as a control for mRNA expression. Relative quantification was carried out via the 2−ΔΔCq method (14 (link)). The primer sequences used are listed in Table SI . RNA extraction, cDNA synthesis and qPCR were performed according to the manufacturer's protocols and these experiments were replicated three times.
5
Quantitative mRNA and miRNA Analysis
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In order to detect the mRNA or miRNA levels, total RNA from lung tissue or A549 cells was extracted using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The RNA concentration was determined via a NanoDrop 2000 micro-spectrophotometer (Thermo Fisher Scientific, Inc.). Next, 1 µg total RNA was utilized to synthesize complementary (c)DNA via a PrimeScript RT Reagent kit (Takara Biotechnology Co., Ltd.). The gDNA elimination reaction was conducted at 42°C for 2 min and reverse transcription was performed at 37°C for 15 min and at 85°C for 5 sec. Subsequently, cDNA was amplified via the TB Green Premix EX Taq II PCR kit (Takara Biotechnology Co., Ltd.). Amplification cycle included an initial 30 sec incubation at 95°C for denaturation, followed by 40 cycles of 5 sec at 95°C for annealing and 30 sec at 60°C for elongation. Small nuclear RNA U6 was utilized for the internal normalization of miR-483-5p, and GAPDH was utilized as a control for mRNA expression. Relative quantification was calculated via the 2−ΔΔCq method (15 (link)). The primer sequences utilized are listed in Table SIII .
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