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  • Title: Quantification of cytokine gene expression using an economical real-time polymerase chain reaction method based on SYBR Green I.
    Author: Ramos-Payán R, Aguilar-Medina M, Estrada-Parra S, González-Y-Merchand JA, Favila-Castillo L, Monroy-Ostria A, Estrada-Garcia IC.
    Journal: Scand J Immunol; 2003 May; 57(5):439-45. PubMed ID: 12753500.
    Abstract:
    Assessment of cytokine expression has become crucial to understand host responses to infections as well as autoimmunity. Several approaches including Northern blot, RNase protection assay and enzyme-linked immunosorbent assay have been used for this purpose, but they are time consuming, labour intense, and relatively large quantity of the samples is usually required. Recently, a technique termed real-time reverse transcriptase-polymerase chain reaction (RT-PCR) has been developed to determine genetic expression with great sensitivity and specificity; however, specialized instrumentation and costly reagents are usually needed. We aimed at using low-cost reagents for real-time PCR. This was achieved by adapting a conventional RT-PCR protocol to the quantitative real-time format, by the addition of the SYBR Green I reagent. We validated the approach by assessing the cytokine gene expression of murine splenocytes upon stimulation with phorbol 12-myristate 12-acetate (PMA)-ionomycin. The results using this technique were compared with those obtained with the well-established gene array method. We conclude that the use of the SYBR Green I reagent during real-time RT-PCR provides a highly specific and sensitive method to quantify cytokine expression with accuracy and no post-PCR manipulation.
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