[Establishment and preliminary application of a quantitative real-time PCR method for detecting the transcriptions of Th1/Th2 cytokines in RAW264.7 cells].

OBJECTIVE

To establish a quantitative real-time PCR method to detect the transcriptions of Th1/Th2 cytokines in RAW264.7 cells.

METHODS

The specific primers were designed according to the sequences of TNF-α, IFN-γ, IL-2, IL-4, IL-6, IL-10 and β-actin in GenBank database. Total RNA was extracted from RAW264.7 cells, and then was reverse-transcriped into cDNA, which was established as a positive standard template of the quantitative real-time PCR method. The established method was used to detect the cytokine transcriptions in RAW264.7 cells infected with Brucella abortus S2308.

RESULTS

Cytokine genes above had a good linear relationship (R(2);≥ 0.982) with the detection limit of 10(2); copies/μL standard samples. The established quantitative real-time PCR method showed high specificity, sensitivity, and single melting peak for every cytokine.

CONCLUSION

The quantitative real-time PCR method for detecting the transcription levels of cytokine Th1 and Th2 of macrophage RAW264.7 cells was established successfully.