TY - JOUR
T1 - Quantification of cytokine gene expression using an economical real-time polymerase chain reaction method based on SYBR® Green I
AU - Ramos-Payán, R.
AU - Aguilar-Medina, M.
AU - Estrada-Parra, S.
AU - González-y-Merchand, J. A.
AU - Favila-Castillo, L.
AU - Monroy-Ostria, A.
AU - Estrada-Garcia, I. C.E.
PY - 2003/5/1
Y1 - 2003/5/1
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0038630795&partnerID=8YFLogxK
U2 - 10.1046/j.1365-3083.2003.01250.x
DO - 10.1046/j.1365-3083.2003.01250.x
M3 - Artículo
SN - 0300-9475
VL - 57
SP - 439
EP - 445
JO - Scandinavian Journal of Immunology
JF - Scandinavian Journal of Immunology
IS - 5
ER -