The cDNA was used as the template for quantitative real-time polymerase chain reaction (qRT-PCR) using the EvaGreen 2X qPCR Master Mix kit (abm, Suzhou, China). Each sample was tested in triplicate, and glyceraldehyde-3-phosphate dehydrogenase was used as a reference gene. Differential gene expression analysis was calculated using the 2−ΔΔCT statistical analysis method. The primers were designed using PrimerPremier.5 software (Table 1 ) and synthesized by Hangzhou Youkang Biotechnology Co., Ltd. (Zhejiang, China).
Evagreen 2x qpcr mastermix kit
Manufactured by Applied Biological Materials
Sourced in Canada, China
EvaGreen 2X qPCR MasterMix kit is a ready-to-use solution for quantitative real-time PCR (qPCR) reactions. It contains EvaGreen, a fluorescent dye that binds to double-stranded DNA, and all the necessary components for PCR amplification.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
All in one rt mastermix kit, by Applied Biological Materials (3 mentions)
Revertaid first strand cdna synthesis kit, by Transgene (1 mentions)
All in one mastermix kit, by Applied Biological Materials (1 mentions)
Abi 7500 fast real time pcr detection system, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Takara Bio (1 mentions)
All in one 5x rt mastermix, by Applied Biological Materials (1 mentions)
Quantstudio 7 flex system, by Thermo Fisher Scientific (1 mentions)
All in one rt mastermix with accurt, by Applied Biological Materials (1 mentions)
Rotor gene q 2plex system, by Qiagen (1 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (1 mentions)
Rotor gene 3000, by Qiagen (1 mentions)
Synergy ht, by Agilent Technologies (1 mentions)
Evagreen 2x qpcr mastermix, by Applied Biological Materials (1 mentions)
Catalase, by Merck Group (1 mentions)
Superoxide dismutase, by Merck Group (1 mentions)
E cadherin, by Merck Group (1 mentions)
N cadherin, by Merck Group (1 mentions)
P erk1 2, by Merck Group (1 mentions)
Mmp 9, by Merck Group (1 mentions)
9 protocols using evagreen 2x qpcr mastermix kit
1
Quantitative Expression Analysis via qRT-PCR
2
Validating Differentially Expressed Genes via qRT-PCR
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To validate the expression of 19 significant DEGs observed in RNA-Seq data, reaction was carried out using EvaGreen 2X qPCR MasterMix Kit (abm, Vancouver, Canada) in a Quanstudio™ 5 Real-Time PCR Intruments (Thermo Fisher Scientific, Inc.). First-strand cDNA was synthesized using the RevertAid™First strand cDNA Synthesis Kit (TransGen Biotech, Beijing, China). DEGs primers were designed using the Primer-Blast (/" https://www.ncbi.nlm.nih.gov/tools/primer-blast/ ) and synthesized commercially (Shuoqing, Kunming, China). Actin were selected as reference genes [121 (link)]. The primers used in qRT-PCR analyses are listed in Table S1 . Amplification reaction mixtures were made of 10 μL of Eva Green 2X qPCR Master Mix, 0.5 μL of each forward and reverse primer (10 mM), and 1 μL of cDNA template, and ddH2O was added to a final volume of 20 μL. The amplification cycling program was as follows: enzyme activation was operated at 95 °C for 10 mins, moreover, 40 cycles of 95 °C for 15 s, 58 °C for 30 s and 72 °C for 30 s. The results were analyzed using the software accompanying the Quanstudio™ 5 Real-Time PCR instruments. The relative expression values were obtained by using the 2-ΔΔCt method [122 (link)].
3
Quantitative PCR Analysis of MyHC Isoforms
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Utilizing the NCBI gene bank, primers of 7 randomly selected DEGs, three different isoforms of MyHC genes (MyHC I, IIA, and IIB) and the house-keeping gene GAPDH were designed by Primer Premier 5 software (Table S1 ). Primers were synthesized by Hangzhou Youkang Biotechnology Co., Ltd. (Zhejiang, China). Total RNA from cells or tissues was used to synthesize cDNA by a 5X All-In-One MasterMix Kit (abm, China). The cDNA was used as a template for quantitative PCR by using the EvaGreen 2X qPCR MasterMix Kit (abm, China). There were three replicates for each sample. The relative expression level of each related gene was calculated using the 2-△△CT statistical analysis method.
4
Gene Expression Quantification by RT-qPCR
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Total RNA of cells and tissues was extracted using TRIzol reagent (Invitrogen, Life Technologies, Carlsbad, CA, USA). Reverse transcription (RT-PCR) was conducted using 5X All-In One RT MasterMix kit (abm, Vancouver, Canada). Quantitative real-time PCR was performed on an ABI 7500 fast real-time PCR Detection System (Life Technologies) using EvaGreen 2X qPCR MasterMix kit (abm, Vancouver, Canada). Primers were designed with Primer 6.0 (ABI, Foster City, CA, USA) (Table S1
5
qRT-PCR Protocol for RPL14 Expression
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In brief, total RNA was extracted from the tissues and cells by Trizol reagent (TaKaRa, Japan). Then, cDNA was synthesized using All-In-One 5X RT MasterMix (Applied Biological Materials Inc., USA). To detect RPL14(eL14) expression, a qRT-PCR assay was performed using an EvaGreen 2X qPCR MasterMix kit (Applied Biological Materials Inc, USA). Primers were synthesized by Sangon Biotech (Shanghai, China). The primer sequences for qRT-PCR were as follows: GAPDH-forward: 5ʹ- TGGGTGTGAACCATGAGAAG-3ʹ, GAPDH- reverse: 5-’ GTGTCGCTGTTGAAGTCAGA-3ʹ. RPL14(eL14)-forward: 5-’TTAAGAAGCTTCAAAAGGCAGC-3ʹ, RPL14(eL14)- reverse: 5-’TTTTGACCCTTCTGAGCTTTTG-3ʹ.
6
Total RNA Extraction and qRT-PCR Analysis
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Total RNA was extracted from the juice sacs using TRIzol™ reagent according to the manufacturer’s instructions (Thermo Scientific). RNA quality was assessed by agarose gel electrophoresis, and 5X All-In-One RT MasterMix with AccuRT (Applied Biological Materials) kit was used for ss/dsDNA digestion and cDNA synthesis. Quantitative RT-PCR was performed on a QuantStudio 7 Flex system (Thermo Scientific, USA) according to the manufacturer’s instruction with EvaGreen 2X qPCR MasterMix kit (Applied Biological Materials, Canada). Elongation factor 1 (Ef1, Cs8g16990) gene was utilized as an internal reference gene (Li et al., 2022 (link)), and the relative expression was calculated with 2-ΔΔCt method. Student’s t-test was performed to determine statistical significance, and ** indicated significant differences at the level of P < 0.01. The primers used for qRT-PCR were listed in Supplementary Table S1
7
Quantitative Gene Expression Analysis Protocol
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Total RNA was extracted from all OS tissues and cells using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Total RNA was dissolved in RNase-free water and the concentration was measured using an Epoch spectrophotometer (ND-1000 spectrophotometer; NanoDrop; Thermo Fisher Scientific, Inc.). cDNA was synthesized using the 5X-All-In-One RT MasterMix kit (Applied Biological Materials, Inc.) according to the manufacturer's protocol, with a Bio-Rad MyCycler system (Bio-Rad Laboratories, Inc.), with the following conditions: 37°C for 15 min and 85°C for 5 sec, followed by cooling to 4°C. The synthesized cDNA was then subjected to qPCR using the EvaGreen 2X qPCR MasterMix kit (Applied Biological Materials, Inc.) on the Rotor-Gene Q 2plex system (Qiagen GmbH). qPCR thermocycling conditions were as follows: Denaturation at 95°C for 10 min, followed by 40 cycles at 95°C for 15 sec, 65°C for 10 sec and 72°C for 15 sec. The standard curves were calculated and the relative quantification of gene expression was assessed. GAPDH expression was used as a standardized internal reference and the 2−ΔΔCq method was used for relative quantification (14 (link)). The sequences of all primers are presented in Table I .
8
RNA Extraction and qRT-PCR Analysis in RAW 264.7 Macrophages
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RAW 264.7 macrophages were treated for 24 h as previously described. By using PureLink® RNA Mini Kit (Invitrogen, Carlsbad, CA, USA), the total RNA content of the cells was determined. Microplate spectrophotometer system (BioTek Synergy HT, Winooski, USA) were used for checking the RNA concentration and purity. 5× All-In-One RT MasterMix kit (Applied Biological Materials Inc. Canada) were used for the synthesized cDNA from 75 ng RNA according to reverse transcription. Real-time PCR (RT-PCR) amplification (Corbett Life Science, Rotor-Gene 3000, Mortlake, Australia) was performed by using EvaGreen 2X qPCR MasterMix (EvaGreen 2X qPCR MasterMix kit, Applied Biological Materials Inc. Canada) of forward and reverse primers (Table 1). Glyceraldehyde-3-phosphate dehydrogenase (GADPH) was used for normalizing quantitative data and the results were calculated by the 2 -ΔΔct method, while the final results were expressed as the fold change compared to control.
9
Comprehensive Molecular Profiling of Cell Culture
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McCoy's 5A and F-12 K media and other reagents for cell culture were purchased from Carlo Erba Reagents (Milan, Italy). The EvaGreen 2X qPCRMaster Mix kit and the primers (Supplementary Table 1) for real time PCR were obtained from Applied Biological Materials Inc. (Canada) and Sigma-Aldrich (Milan, Italy). The primary and secondary antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA) and Sigma-Aldrich (Milan, Italy).
The following antibodies were used: Nrf2, superoxide dismutase (SOD), catalase, heme oxygenase 1 (HO-1), NF-κB, phosphorylated inhibitor of kappa B (p-IκBα), p53, caspase-3, cleaved-poly (ADP-ribose) polymerase (c-PARP), EGFR, human epidermal growth factor receptor 2 (HER2), p-Akt, p-mammalian target of rapamycin (p-mTOR), p-p38MAPK, p-extracellular-signal-regulated kinase 1/2 (p-Erk1/2), matrix metalloproteinases 2 (MMP-2), MMP-9, E-cadherin, N-cadherin, β-catenin and glyceraldehyde-3-phosphate dehydrogenase (GADPH). All other chemicals were purchased from Sigma-Aldrich (Milan, Italy). MH samples inventing from New Zealand were imported to Italy by EfitSrl and kept at 4°C until analysis.
The following antibodies were used: Nrf2, superoxide dismutase (SOD), catalase, heme oxygenase 1 (HO-1), NF-κB, phosphorylated inhibitor of kappa B (p-IκBα), p53, caspase-3, cleaved-poly (ADP-ribose) polymerase (c-PARP), EGFR, human epidermal growth factor receptor 2 (HER2), p-Akt, p-mammalian target of rapamycin (p-mTOR), p-p38MAPK, p-extracellular-signal-regulated kinase 1/2 (p-Erk1/2), matrix metalloproteinases 2 (MMP-2), MMP-9, E-cadherin, N-cadherin, β-catenin and glyceraldehyde-3-phosphate dehydrogenase (GADPH). All other chemicals were purchased from Sigma-Aldrich (Milan, Italy). MH samples inventing from New Zealand were imported to Italy by EfitSrl and kept at 4°C until analysis.
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