The qRT-PCR was performed as described by Sun et al.49 (link). Briefly, total RNA was isolated and reverse transcribed as described above. The cDNA was amplified using TaqMan Universal PCR master mix (Roche Applied Science) and a LightCycle® 96 detection system (Roche Applied Science). The amplification of the target genes was normalized using the amplification levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an endogenous control. The efficiency of the PCR was tested by amplification of the target from serially diluted cDNA generated from the reverse transcription of a stock set of human RNA. The data analysis and calculations were performed using the 2−ΔΔCT comparative method, as described by the manufacturer. Gene expression is shown as the fold induction of a gene measured in IL-1-treated samples relative to samples cultured with medium. Same primes were used as described in RT-PCR.
Lightcycle 96 detection system
Manufactured by Roche
The LightCycle® 96 detection system is a real-time PCR (Polymerase Chain Reaction) instrument designed for accurate and reliable nucleic acid quantification. It features a 96-well format and can perform thermal cycling and fluorescence detection to enable precise analysis of DNA and RNA samples.
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Lab products found in correlation
2 protocols using lightcycle 96 detection system
1
Quantitative Real-Time PCR Protocol
2
Quantitative RT-PCR Assay for Gene Expression
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The qRT-PCR assay was performed as described ]. Briefly, total RNA was isolated and reverse transcribed as described above. The cDNA templates were amplified using TaqMan Universal PCR master mix (Roche Applied Science) and a LightCycle 96 detection system (Roche Applied Science). The amplification of the target genes was normalized to the levels of glyceraldehyde-3phosphate dehydrogenase (GAPDH) as an endogenous control. The efficiency of PCR was tested by amplifying the target from serially diluted cDNA samples generated from the reverse transcription of a stock set of human RNAs. The data were analyzed and calculations were performed using the 2 -ΔΔCT comparative method, as described by the manufacturer. Gene expression is presented as the fold induction of a gene measured in IL-1-and/or MDP-treated samples relative to samples cultured with medium. The same primers were used as described for RT-PCR.
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