title: Quantification of cytokine gene expression using an economical real-time polymerase chain reaction method based on SYBR Green I.
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.
authors.affiliation: Departamento de Inmunología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, México D F, México.
authors.forename: R
authors.initials: R
authors.lastname: Ramos-Payán
authors.forename: M
authors.initials: M
authors.lastname: Aguilar-Medina
authors.forename: S
authors.initials: S
authors.lastname: Estrada-Parra
authors.forename: J A
authors.initials: JA
authors.lastname: González-Y-Merchand
authors.forename: L
authors.initials: L
authors.lastname: Favila-Castillo
authors.forename: A
authors.initials: A
authors.lastname: Monroy-Ostria
authors.forename: I C E
authors.initials: IC
authors.lastname: Estrada-Garcia
mesh_terms: D000199:Actins / Q000096:biosynthesis / Q000235:genetics; D000818:Animals; D052160:Benzothiazoles; D003199:Computer Systems / Q000191:economics; D003362:Cost-Benefit Analysis; D003365:Costs and Cost Analysis; D016207:Cytokines / Q000096:biosynthesis* / Q000235:genetics; D003959:Diamines; D005260:Female; D005456:Fluorescent Dyes / Q000032:analysis*; D020869:Gene Expression Profiling / Q000191:economics / Q000379:methods*; D007202:Indicators and Reagents / Q000191:economics; D007371:Interferon-gamma / Q000096:biosynthesis / Q000235:genetics; D018664:Interleukin-12 / Q000096:biosynthesis / Q000235:genetics; D053762:Interleukin-12 Subunit p40; D007376:Interleukin-2 / Q000096:biosynthesis / Q000235:genetics; D015759:Ionomycin / Q000494:pharmacology; D008213:Lymphocyte Activation / Q000187:drug effects; D051379:Mice; D008807:Mice, Inbred BALB C; D020411:Oligonucleotide Array Sequence Analysis; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D021122:Protein Subunits / Q000096:biosynthesis / Q000235:genetics; D011804:Quinolines; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis*; D012680:Sensitivity and Specificity; D013154:Spleen / Q000166:cytology; D013601:T-Lymphocytes / Q000187:drug effects / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D016212:Transforming Growth Factor beta / Q000096:biosynthesis / Q000235:genetics
chemical_list: D000199:Actins; D052160:Benzothiazoles; D016207:Cytokines; D003959:Diamines; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D053762:Interleukin-12 Subunit p40; D007376:Interleukin-2; D009930:Organic Chemicals; D021122:Protein Subunits; D011804:Quinolines; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; C098022:SYBR Green I; D018664:Interleukin-12; D015759:Ionomycin; D007371:Interferon-gamma; D013755:Tetradecanoylphorbol Acetate