Total RNA extraction and cDNA synthesis were performed using TRIzol (Thermo Fisher, #15596026) and the Evo M-MLV RT Premix kit (Accurate Biology, AG11706) according to the manufacturer’s instructions. Gene transcription levels were determined using the SYBR Green Pro Taq HS Premix kit (Accurate Biology, AG11701) with the primer sequences (Tsingke Biotechnology). CDKN1A primer sequences are as forward primer 5′-CGATGGAACTTCGAC TTTGTCA-3′ and reverse primer 5′-GCACAAGGGTACAAGACAGTG-3′. PUMA primer sequences are as: forward primer 5′-GCCAGATTTGTGAGACAAGAGG-3′ and reverse primer 5′-CAGGCACCTAATTGGGCTC-3′. NOXA primer sequences are as: forward primer 5′-ACCAAGCCGGATTTGCGATT-3′ and reverse primer 5′-ACTTGCACTTGTTCCTCGTGG-3′. FANCD2 primer sequences are as: forward primer 5′-GTTCGCCAGTTG GTGATGGAT-3′ and reverse primer 5′-GGGAAGCCTGTAACCGTGAT-3′.
Sybr green pro taq hs premix kit
Manufactured by Accurate Biology
Sourced in China
The SYBR Green Pro Taq HS Premix kit is a reagent used for real-time polymerase chain reaction (qPCR) analysis. It contains a premixed solution of Taq DNA polymerase, SYBR Green I dye, and necessary buffers and reagents for efficient and sensitive DNA amplification and detection.
Automatically generated - may contain errors
Lab products found in correlation
Evo m mlv rt premix kit, by Accurate Biology (2 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Cfx96 qpcr system, by Bio-Rad (1 mentions)
Rnaiso plus reagent, by Takara Bio (1 mentions)
Nanodrop 2000c spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Steadypure universal rna extraction kit, by Accurate Biology (1 mentions)
Tprofessional thermocycler, by Analytik Jena (1 mentions)
Evo m mlv reverse transcription kit, by Accurate Biology (1 mentions)
Abi viia7 pcr machine, by Thermo Fisher Scientific (1 mentions)
Evo m mlv rt kit, by Accurate Biology (1 mentions)
Cfx96 system, by Bio-Rad (1 mentions)
Nanodrop 3300 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Hiscript 3 1st strand cdna synthesis kit gdna wiper, by Vazyme (1 mentions)
6 protocols using sybr green pro taq hs premix kit
1
Quantitative RNA Expression Analysis
2
Validating RNA-seq Transcriptome with RT-qPCR
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To validate the results obtained from the transcriptome, we selected 10 upregulated genes and 11 downregulated genes of interest for two-step RT-qPCR verification with the same RNA-seq samples. Reverse transcription was performed using the Evo M-MLV RT Master Mix for qPCR kit (AG11706, Accurate Biology, China), and real-time qPCR was carried out using the SYBR® Green Pro Taq HS Premix kit (AG11701, Accurate Biology, China). The Bio-Rad CFX96 qPCR System was used for RT-qPCR, and using the reaction as follows: initial denaturation at 95°C for 30 s, followed by 40 cycles of reaction at 95°C for 5 s and 60°C for 30 s. The melting curve was generated at the end of the cycle to confirm the specificity of the amplification product. Each PCR reaction was repeated three times. Gene expression was calculated and compared using the 2−ΔΔCt method, with 16s rRNA serving as an endogenous reference. The primers used for the experiment are listed in Supplementary Table 1 .
3
Rat Uterine RNA Isolation and qPCR Analysis
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Total RNA was isolated from rat uterine tissue using the RNAiso Plus Reagent (Takara, Japan) following the manufacture’s instruction, and quantified using a NanoDrop 2000c Spectrophotometer (Thermo Fisher Scientific). Reverse transcription was performed using the Evo M-MLV RT Premix kit (Accurate Biotechnology, China), and cDNA was input into qPCR system with the SYBR Green Pro Taq HS Premix Kit (Accurate Biotechnology). Reaction of qPCR was run in technical duplicates on an ABI ViiA 7 platform (Thermo Fisher Scientific). The primer sequences for RT-qPCR are shown in Table 1 . Specificity of qPCR reaction was confirmed by single peak in melting curve. The relative expression of each gene was normalized to GAPDH.
4
Liver mRNA Expression Analysis of BSA@Cu2−xS NPs
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The changes in mRNA expression were evaluated by RT-qPCR in the liver tissue from BSA@Cu2−xS NPs and the corresponding recovery groups. Total RNA was extracted from the livers of rats treated with 8 mg/kg LNPs and SNPs at the end of the dosing period (Day 14) and recovery period (Day 42) using a SteadyPure Universal RNA Extraction Kit (Accurate Biotechnology, Hunan, China) according to the supplier’s instructions. RNA was reverse transcribed into cDNA using an Evo M-MLV reverse transcription kit (Accurate Biotechnology, Changsha, China) carried out by a gradient PCR machine (Tprofessional Thermocycler, Biometra, Germany). The reaction program was set up as follows: 37 °C for 15 min, 85 °C for 5 s, and cool down to 4 °C. Real-time quantitative PCR (RT-qPCR) was performed using a SYBR Green Pro Taq HS premix kit (Accurate Biotechnology, Changsha, China) on an ABI ViiA7 PCR machine (Applied Biosystems, Life Technologies, USA). The thermocycler parameters were set up as follows: 95 °C for 30 s, 40 cycles of 95 °C for 5 s and 60 °C for 30 s. Ct values were used to analyze the difference between the dosing group and the control group. All primers used in this study are listed in Additional file 1 : Table S1.
5
ccRCC Prognosis-related Gene Expression
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Paraffin tissue blocks and clinical data from 22 ccRCC patients were collected from Shaanxi Provincial People’s Hospital. RT-PCR was employed to detect the expression of the prognosis-related genes and activated dendritic cell markers. A DNA/RNA extraction kit (AmoyDx, Xiamen, China) was used to extract RNA from the tissue paraffin blocks. The Evo M-MLV RT kit and SYBR® Green Premix Pro Taq HS kit (Accurate Biology, Changsha, China) were used to conduct two-step fluorescence quantitative PCR. The Cq value was employed to calculate the gene expression based on 2−ΔΔCt. The internal reference gene was GAPDH.
6
Quantitative Gene Expression Analysis of Left Ventricular Samples
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RNA was extracted separately for each biological replicate from the MI area of left ventricular samples using Trizol Reagent (Megan Biotech, Guangzhou, China) in accordance with the manufacturer’s instructions. The concentration and quality of the total RNA samples were measured using the NanoDrop 3300 Spectrophotometer (ThermoFisher Scientific). RNA integrity was assessed by electrophoresis using denaturing agarose gels. High-quality RNA samples were used to synthesize the cDNA via the HiScript® III 1st Strand cDNA Synthesis Kit (+ gDNA wiper) (Vazyme, Nanjing, Jiangsu, China) in accordance with the manufacturer’s protocol. Finally, the qRT-PCR was performed using the SYBR® Green Premix Pro Taq HS Kit (Accurate Biology, Changsha, Hunan, China) with gene-specific primers in a Bio-Rad CFX96 system (Bio-Rad, Hercules, California, USA). RNA relative expression levels were calculated using the 2−ΔΔCT method against the internal control glyceraldehyde-3-phosphate dehydrogenase.
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