Establishment of relative quantification in real time reverse transcription polymerase chain reaction to measure cytokine expression

Objective To establish relative quantification in real time reverse transcription polymerase chain reaction to measure cytokine expression.Methods TNFα and GAPDH were used as target gene and internal reference respectively. Two gene fragment were cloned, vectors were purified and quantificated. Standard curves were established using a series dilution of quantificated plasmids to measure the amplification efficiency of TNFα and GAPDH. By changing reaction conditions, the amplification efficiency of two gene were nearly 100%. Ct value of TNFα and GAPDH were measured in 14 samples stimuteneously.Results The method can detected as low as 10 3 copies with the linear range was 10 3～10 9 copies, the intra assay and interassay variation was 1% and 8% respectively. TNFα increased 8 6～9 8 fold with the average 9 2 relative to untreated control group analyzed by the 2 -ΔΔCt methods. Conclusions The method we established has fine sensitivity and reproducibility and the data analysis was simple and reliable and can be apply to any genes.