cDNA construction was performed using SuperPrep Cell Lysis & RT Kit for qPCR (Toyobo Co., Japan) according to the manufacturer's instructions. TaqMan PCR reagents for DR4 (Hs00269492), DR5 (Hs00366278) and SOCS3 (Hs02330328) were purchased from ABI (Applied Biosystems, CA). Quantitative real-time PCR was carried out using the TaqMan Master Mix Reagents Kit protocol on a StepOne real-time PCR System (Applied Biosystems, CA). The data were standardized against β-actin gene expression using TaqMan β-actin control reagent (Applied Biosystems, CA).
Taqman master mix reagents kit
Manufactured by Thermo Fisher Scientific
The TaqMan Master Mix reagents kit is a set of pre-formulated reagents designed for real-time PCR (polymerase chain reaction) assays. The kit contains all the necessary components, including DNA polymerase, dNTPs, and buffer, to perform qPCR (quantitative PCR) experiments. The reagents are optimized for sensitivity and specificity to enable accurate and reliable gene expression analysis and quantification.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (3 mentions)
Iscript cdna synthesis kit, by Bio-Rad (3 mentions)
Stepone real time pcr system, by Thermo Fisher Scientific (2 mentions)
Real time pcr system, by Bio-Rad (2 mentions)
Superprep cell lysis rt kit for qpcr, by Toyobo (1 mentions)
Abi 7300 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Pre developed taqman assay reagents, by Thermo Fisher Scientific (1 mentions)
5 protocols using taqman master mix reagents kit
1
Quantitative Analysis of Death Receptor and SOCS3 Expression
2
Quantitative Analysis of Gene Expression
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Total RNA of cells was isolated by Rneasy Mini Kit (QIAGEN). At least 1 μg of total RNA was used for cDNA synthesis by the iSCRIPT cDNA Synthesis Kit (Bio-Rad) according to the manufacturer’s instructions. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed in an BioRad real-time PCR system. All primers and probes used for RT-qPCR were purchased from Applied Biosystems. RNA RT-PCR samples were prepared by using the TaqMan Master Mix reagents kit (Applied Biosystems). Gene expression analysis were calculated with ΔΔCT method to determine the quantification of gene expression. We normalized the target gene expression in the test samples to levels of the endogenous reference, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and reported them as the fold-difference relative to GAPDH gene expression in untreated baseline controls.64 (link) All assays were performed with at least triplicate samples, and experiments were repeated more than two times.
3
Quantitative Analysis of Gene Expression
4
RNA Extraction and RT-qPCR Analysis
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We used the RNeasy Mini Kit (QIAGEN, Hilden, Germany) to isolate total RNA (Ribonucleic acid) from cells. At least 1 µg of total RNA was used for cDNA synthesis by the iSCRIPT cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) in accordance with the manufacturer’s instructions. A real-time quantitative polymerase chain reaction (RT-qPCR) was performed in an ABI 7300 Real-Time PCR system (Applied Biosystems, Foster, CA). All primers and probes used for the RT-qPCR were purchased from Applied Biosystems. RNA RT-PCR samples were prepared by using the TaqMan Master Mix reagents kit (Applied Biosystems). Gene expression analyses were calculated with the ΔΔCT method to determine the quantification of gene expression. We normalized the target gene expression in the test samples to the levels of the endogenous reference, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and reported them as the fold difference relative to GAPDH gene expression in untreated baseline controls [42 (link)]. All assays were performed with triplicated samples, and experiments were repeated more than two times.
5
Quantitative Gene Expression Analysis by TaqMan PCR
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TaqMan PCR reagents for the 5 genes with the highest or lowest expression genes on the microarray were purchased from ABI (Applied Biosystems, CA), and applied according to the TaqMan Master Mix reagents kit protocol. The reactions were incubated for 2 min at 50°C, followed by denaturation for 10 min at 95°C. The reactions were run for 40 cycles of denaturation for 15 sec at 95°C, and an extension for 1 min at 60°C per cycle using a StepOne real-time PCR System (Applied Biosystems, CA). The data were standardized against beta-actin gene expression using Pre-Developed TaqMan Assay Reagents (Applied Biosystems, CA).
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