An ABI 7500 Real-time PCR system (Applied Biosystems, USA) was used as a fluorescence quantification platform in this study. The reaction system was 10 μl, including 5 μl TaqMan Universal Master Mix II with uracil-N-glycosylase (purchased from Applied Biosystems, USA), 0.4 μl sense primer (ASFV-For), 0.4 μl anti-sense primer (ASFV-Back), 0.4 μl of probe, 1.8-μl nuclease-free water (Promega, USA), and 2 μl of template. The optimal concentrations of primers and the probe were then measured when the ASFV pFastBacI-p72 plasmid was 1 × 108 copies/ml. Primers with optimal concentration were determined by 12.5, 25, 50, and 100 μM; meanwhile, the probe with optimal concentration was selected by 1.25, 2.5, 5, and 10 μM. The qPCR program was carried out as follows: initial denaturation at 95°C for 10 min, and at 95°C for 15 s, cycling 40 times, and at 60°C holding for 45 s. Negative and positive controls were set at the same time in a run.
Taqman universal master mix 2 with uracil n glycosylase
Manufactured by Thermo Fisher Scientific
Sourced in United States
TaqMan Universal Master Mix II with uracil-N-glycosylase is a ready-to-use solution for real-time PCR. It contains all the necessary components, including a thermostable DNA polymerase, dNTPs, magnesium, and a buffer system optimized for TaqMan probe-based detection.
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Lab products found in correlation
Rneasy mini kit, by Qiagen (2 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (2 mentions)
Abi 7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Nuclease free water, by Promega (1 mentions)
Nanodrop nd 1000, by Thermo Fisher Scientific (1 mentions)
Nucleospin rna, by Macherey-Nagel (1 mentions)
Taqman mgb probe, by Thermo Fisher Scientific (1 mentions)
Applied biosystems high capacity reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
7500 fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Qiazol lysis reagent, by Qiagen (1 mentions)
Stepone software version 2, by Thermo Fisher Scientific (1 mentions)
Multiscribe reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Stepone software, by Thermo Fisher Scientific (1 mentions)
Cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
4 protocols using taqman universal master mix 2 with uracil n glycosylase
1
Quantitative Real-Time PCR for ASFV Detection
2
Quantitative Gene Expression Analysis
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Total RNA was extracted from PBMC and liver using NucleoSpin® RNA (Macherey Nagel, Düren, Germany), as described by the manufacturer. The RNA concentration was determined using NanoDrop ND-1000 (NanoDrop Technologies, Rockland, DE, USA) spectrophotometer. The complementary DNA (cDNA) synthesis was carried out using Applied Biosystems High-Capacity Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA), as described by the manufacturer.
Real-time reverse-transcribed PCR was carried out in a 7500 Fast real-time PCR system (Applied Biosystems, Foster City, CA, USA) using custom-designed TaqMan MGB probes on targeted genes Interleukin-1beta (IL-1β), Interleukin-6 (IL-6), Tumor Necrosis Factor-alpha (TNF-α), and Intercellular Adhesion Molecule (ICAM) in PBMC. The bovine RPS9 and GAPDH were used as endogenous controls, also called housekeeping genes (Table 2 ). Reaction mixture included 2 μL of cDNA, 10 µL of TaqMan® Universal Master Mix II with Uracil-N-Glycosylase (Applied Biosystems, Foster City, CA, USA), 1 μL of Applied Biosystems 20X custom primer probe mixture (Applied Biosystems, Foster City, CA, USA), and 7 μL of RNase-free water.
Real-time reverse-transcribed PCR was carried out in a 7500 Fast real-time PCR system (Applied Biosystems, Foster City, CA, USA) using custom-designed TaqMan MGB probes on targeted genes Interleukin-1beta (IL-1β), Interleukin-6 (IL-6), Tumor Necrosis Factor-alpha (TNF-α), and Intercellular Adhesion Molecule (ICAM) in PBMC. The bovine RPS9 and GAPDH were used as endogenous controls, also called housekeeping genes (
3
Synovial Transcriptional Profile in Osteoarthritis
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Synovial tissue specimens from ankle joints were harvested at 10, 14 and 16 weeks of age (n = 5 for each group). Total RNA was extracted using QIAzol Lysis Reagent (Qiagen, Hilden, Germany) and an RNeasy Mini Kit (Qiagen, Hilden, Germany). Synthesis of cDNA from total RNA was carried out using RT buffer, RT random primers, dNTP mix, and Multiscribe reverse transcriptase (Applied Biosystems, Foster city, CA, USA). A total of 9 μL cDNA diluted 1:9 was added to 10 μL Taqman Universal Master Mix II with Uracil N-glycosylase (Applied Biosystems, Foster City, CA, USA). Real-time amplification of the genes was performed using 1 μL ready-to-use Taqman Gene Expression Assays (Applied Biosystems) for Il6, Tnf, IL17, and gapdh as an endogenous control (assay IDs: Mm00446190_m1, Mm00443260_g1, Mm00439618_m1, and Mm99999915_g1). Relative gene expression data were analyzed using the delta-delta-Ct method with PCR-efficiency correction using StepOne software version 2.2.2 (Applied Biosystems), as previously described in the literature [32 (link)].
4
Quantifying Bone Callus Gene Expression
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The callus specimens were harvested at 7, 10, and 14 days after surgery. Callus specimens were collected from six mice in each group on days 7, 10, and 14, for a total of 36 mice in both groups were used for the RT-PCR analysis. Muscles and original bone were separated from the calluses. Quantitative RT-PCR was performed as previously described (Izumiyama et al. 2019) . The extraction of total RNA was performed using TRIzol (Invitrogen Corp., Carlsbad, CA, USA) and RNeasy Mini Kit (Qiagen, Hilden, Germany). Synthesis of cDNA from the total RNA was performed using cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA). Real-time amplification of the target genes was performed using Taqman Universal Master Mix II with Uracil-N glycosylase and ready-to-use Taqman Gene Expression Assays (Applied Biosystems) for collagens (Col1a1, Mm00801666_g1; Col2a1, Mm01309565_m1; Col10a1, Mm00487241_m1), runt-related transcription factor 2 (Runx2, Mm00501584_ m1), dickkopf-related protein 1 (Dkk1, Mm00438422_m1), acid phosphatase 5 tartrate resistant (Acp5, Mm00475698_ m1), and Glyceraldehyde-3-phosphate dehydrogenase (Gapdh, Mm99999915_g1) as an endogenous control. Relative gene expression data were calculated using the delta-delta-Ct method with PCR-efficiency correction using StepOne software (version 2.2.2; Applied Biosystems).
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