Relative tumour necrosis factor-α (TNF-α), nuclear factor kappa beta (NFK-ß), and interleukin-6 (IL-6) messenger RNA (mRNA) expression analyses were performed using StepOne Plus Real-Time polymerase chain reaction (PCR) system technology (Applied Biosystems, Foster City, CA, USA) using synthesised cDNA from rat kidney RNA. A quantitative PCR was run using a TaqMan probe mix based on TaqMan probe-based technology (Applied Biosystems, Foster City, CA, USA). Real-time PCR was performed using primers generated for rat TNF-α Rn00562055_m1, rat NFK-ß Rn01399583_m1, rat IL-6 Rn01410330_m1, and rat β-actin Rn00667869_m1. The results are expressed as relative-fold, compared with control animals. The expression data for β-actin in each tissue were used as the endogenous control. Each determination was performed in triplicate for each tissue in a 96-well optical plate for both targets, using 9-μL cDNA (100 ng), 1 μL of Primer Perfect Probe mix, and 10 μL of QuantiTect Probe PCR Master Mix (Qiagen) in each 20-μL reaction. The plates were heated for 2 min at 50 °C and then 10 min at 95 °C. Subsequently, 40 cycles of 15 s at 94 °C and cycles of 60 s at 60 °C were conducted. All data are expressed as the fold-change in expression compared with the expression in other animal groups, using the 2-delta-delta Ct (2-ΔΔCt) method [13 (link), 14 (link)].
Taqman probe mix
Manufactured by Thermo Fisher Scientific
Sourced in United States
TaqMan probe mix is a ready-to-use solution designed for qPCR (quantitative Polymerase Chain Reaction) experiments. It contains all the necessary components, including a thermostable DNA polymerase, dNTPs, and a fluorescent probe, to enable sensitive and specific detection of target DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Quantitect probe pcr master mix, by Qiagen (2 mentions)
Taqman probe based technology, by Thermo Fisher Scientific (2 mentions)
Primer perfect probe mix, by Qiagen (2 mentions)
Rneasy kit, by Qiagen (2 mentions)
Steponeplus real time polymerase chain reaction, by Thermo Fisher Scientific (1 mentions)
Steponeplus real time pcr system technology, by Thermo Fisher Scientific (1 mentions)
First strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
7900ht pcr cycler, by Thermo Fisher Scientific (1 mentions)
Qx200 droplet digital system, by Bio-Rad (1 mentions)
Quantasoft, by Bio-Rad (1 mentions)
Qx200 droplet digital pcr system, by Bio-Rad (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Nucleospin rna kit, by Takara Bio (1 mentions)
Stepone real time pcr system, by Thermo Fisher Scientific (1 mentions)
Qiaamp circulating nucleic acid kit, by Qiagen (1 mentions)
Qx200 droplet reader, by Bio-Rad (1 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (1 mentions)
6 protocols using taqman probe mix
1
Quantitative PCR Analysis of Inflammatory Markers
2
Quantifying TNF-α and NF-κB Expression in Rat Kidney
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Relative tumor necrosis factor (TNF)-α and nuclear factor (NF)-κB expression analyses were performed with StepOne Plus Real Time PCR System technology (Applied Biosystems, Foster City, CA) using synthesized cDNA from rat kidney RNA. A quantitative polymerase chain reaction (qPCR) was run using a TaqMan probe mix based on TaqMan probe-based technology (Applied Biosystems). A real-time PCR was performed using primers generated for rat TNF-α Rn00562055_m1, rat NF-κB1 Rn01399583_m1, and rat β-actin Rn00667869_m1. The results are expressed as relative-fold, compared with control animals. The expression data for β-actin in each tissue were used as the endogenous control. Each determination was performed in triplicate for each tissue in a 96-well optical plate for both targets, using 9 μL of cDNA (100 ng), 1 μL of Primer Perfect Probe mix, and 10 μL of QuantiTect Probe PCR Master mix (Qiagen) in each 20-μL reaction. The plates were heated for 2 min at 50 °C and then 10 min at 95 °C. Subsequently, 40 cycles of 15 s at 94 °C and cycles of 60 s at 60 °C were carried out. All data are expressed as the fold-change in expression compared to the expression in other animal groups, using the 2-delta-delta Ct (2-ΔΔCt) method.18 (link)
3
Quantifying NIS Expression in HDACi-Treated Cells
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After the cells were treated with HDACi for 48 hours, RNA was extracted using RNeasy kit (QIAGEN, USA). cDNAs were synthesized using the first strand cDNA synthesis kit (Invitrogen, USA). Quantitative real-time PCR was performed using Taqman probe mix on the 7900HT PCR cycler (Applied Biosystems, USA). The Taqman probes for human NIS and GAPDH with assay ids Hs00166567_m1 and Hs02758991_g1 respectively were used (Applied Biosystems, USA). Triplicate samples were run for each sample. The comparative ΔΔCt method was used to calculate relative gene expression.
4
Droplet Digital PCR for EGFR Mutation Analysis
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Each PCR sample was partitioned into 20 000 droplets and analyzed using a QX200 Droplet Digital PCR system (Bio‐Rad Laboratories). Following amplification, each droplet was scored as positive or negative after the detection of the fluorescence signal emitted by the target sequence. Poisson statistical analysis was performed for the absolute quantification of the target sequence. To analyze EGFR mutations, in addition to a reference assay, TaqMan EGFR T790M mutation assay, EGFR Exon 19 deletions assay, EGFR L858R mutation assay, and EGFR C797S mutation assay (Invitrogen Life Technologies) were performed. In brief, 20 μL of a ddPCR reaction mix containing 2X Master Mix (Bio‐Rad Laboratories), 20X primer, and TaqMan Probe mix (Applied Biosystems Life Technologies) in addition to the DNA template was prepared and subjected to emulsification using the QX200 droplet generator. The emulsified samples were then transferred into 96‐well plates for PCR, and the PCR products were loaded into a droplet reader (QX200 Droplet Digital System; Bio Rad Laboratories) for analysis of mutations through QuantaSoft (version 1.7.4.0917; Bio‐Rad Laboratories) The result was interpreted as positive if the mutant copy number was ≥3 in the ddPCR assays. Fractional abundance (FA), which indicates the abundance of mutant DNA alleles in a wild‐type background, was also evaluated.19
5
Quantitative RT-PCR of Inflammatory Markers
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Total RNA was extracted from cultured cells using a Nucleospin RNA Kit (TaKaRa Bio, Shiga, Japan) and from tissues with an RNeasy Kit (Qiagen, Hilden, Germany). Complementary DNA was prepared using a PrimeScript RT Reagent Kit (TaKaRa Bio). Quantitative real‐time PCR was performed using TaqMan Probe Mix (Applied Biosystems, Foster City, CA, USA) and the StepOne™ Real‐Time PCR system (Applied Biosystems). The reactions were cycled as follows: initial incubation at 95°C for 20 s, followed by 50 cycles of 95°C for 1 s and 60°C for 20 s. Predesigned primer/probe sets were as follows: β‐actin, Mm00607939_s1; IL‐6, Mm99999064_m1; IL‐1β, Mm00434228_m1; MCP‐1, Mm00441242_m1; IP‐10, Mm00445235_m1; TNF, Mm00443260_g1; HP, Mm00516884_g1; SAA1, Mm00656927_g1. For each target, mRNA expression levels were calculated relative to those of β‐actin in accordance with the manufacturer’s protocol.
6
ddPCR Validation of EGFR T790M
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Before submission, PT samples were subjected to between-run and within-run validation using the ddPCR platform. Briefly, blood samples were centrifuged at 1500 g for 10 minutes to isolate plasma for cfDNA extraction using QIAamp Circulating Nucleic Acid Kit (Qiagen, Hilden, Germany), according to the manufacturer's protocols. The quality and quantity of extracted cfDNA were assessed using Qubit fluorometer (Thermo Fisher Scientific). A total ddPCR reaction mixture of 20 μL containing 2× Master Mix (Bio-Rad Laboratories, Inc.), 20× primers, and TaqMan Probe mix (Applied Biosystems Life Technologies), nuclease-free water and DNA template, and 70 μL of droplet generation oil were subsequently loaded into the droplet generator cartridge. The cartridge was then placed into the droplet generator for emulsification to produce individual droplets. The emulsified sample was then transferred to a 96-well PCR plate for PCR amplification, after which the plate was placed on a QX200 droplet reader (Bio-Rad) to analyze the droplets in each well. The PCR data were further analyzed using QX200 software version 1.7.4.0917 (Bio-Rad Laboratories, Inc.). The frequencies of EGFR T790M detected in the PT samples (sample A, B, C, and D) are summarized in Table 1.
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