Total RNA was isolated using TRIzol reagent (Life Technologies). Reverse transcription was performed using the High capacity cDNA Reverse Transcription Kit (Life Technologies), according to the manufacturer’s protocol. The gene expression levels were quantified by TaqMan-based real-time PCR reactions (Life Technologies). The used TaqMan probes are listed in Supplementary Table 1 . The expression levels were normalized to RPLP0. Results were calculated using the ΔΔCt method and presented as fold increase relative to control.
Taqman based real time pcr reactions
Manufactured by Thermo Fisher Scientific
TaqMan-based real-time PCR reactions are a method for detecting and quantifying specific DNA sequences. This technology uses fluorescent probes to monitor the amplification of target DNA sequences during the PCR process. The core function of this product is to provide a sensitive and reliable way to measure gene expression levels or detect the presence of specific genetic targets.
Automatically generated - may contain errors
3 protocols using taqman based real time pcr reactions
1
Gene Expression Quantification via RT-qPCR
2
Measuring mRNA Expression by RT-qPCR
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Total RNA from the cell culture was isolated using TriZol (Life Technologies). Reverse transcription was performed using the QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany). The mRNA expression was determined by TaqMan-based real-time PCR reactions (Life Technologies). The following TaqMan probes were used: Hs01029144_m1 (human ALPL) and 4326315E (human β-actin). The expression levels were normalized to β-actin. Results were calculated using the ΔΔCt method, and presented as fold increase relative to control.
3
Quantification of Inflammatory Markers
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Total RNA from the cell culture was isolated using TriZol (Life Technologies), and reverse transcription was performed using the QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany). The mRNA expression was determined by TaqMan-based real-time PCR reactions (Life Technologies). The following TaqMan probes were used: Hs99999902_m1 (human RPLP0), Mm00725448_s1 (mouse RPLP0), Hs00981511_m1 (human PARP14), Mm00520984_m1 (mouse PARP14), Hs00967084_m1 (human PARP9), Mm00518778_m1 (mouse PARP9), Hs00174128_m1 (human TNF), Mm00443258_m1 (mouse TNF), Hs00174097_m1 (human IL-1β), Mm01336189_m1 (mouse 1L1β), Mm00475988_m1 (mouse ARG1), Hs00267207_m1 (human MRC1), Mm00485148_m1 (mouse MRC1), Mm00440502_m1 (mouse NOS2) and Hs00234140_m1 (human CCL2). The expression levels were normalized to RPLP0. Results were calculated using the Delta-Delta Ct method, and presented as arbitrary unit.
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