Quantification of cytokine gene expression using an economical real-time polymerase chain reaction method based on SYBR Green I.
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.
Ramos-Payán
R
R
Departamento de Inmunología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, México D F, México.
Aguilar-Medina
M
M
Estrada-Parra
S
S
González-Y-Merchand
J A
JA
Favila-Castillo
L
L
Monroy-Ostria
A
A
Estrada-Garcia
I C E
IC
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
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