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.
Publication types
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Evaluation Study
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Research Support, Non-U.S. Gov't
MeSH terms
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Actins / biosynthesis
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Actins / genetics
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Animals
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Benzothiazoles
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Computer Systems / economics
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Cost-Benefit Analysis
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Costs and Cost Analysis
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Cytokines / biosynthesis*
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Cytokines / genetics
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Diamines
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Female
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Fluorescent Dyes / analysis*
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Gene Expression Profiling / economics
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Gene Expression Profiling / methods*
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Indicators and Reagents / economics
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Interferon-gamma / biosynthesis
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Interferon-gamma / genetics
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Interleukin-12 / biosynthesis
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Interleukin-12 / genetics
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Interleukin-12 Subunit p40
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Interleukin-2 / biosynthesis
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Interleukin-2 / genetics
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Ionomycin / pharmacology
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Lymphocyte Activation / drug effects
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Mice
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Mice, Inbred BALB C
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Oligonucleotide Array Sequence Analysis
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Organic Chemicals*
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Polymerase Chain Reaction / economics
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Polymerase Chain Reaction / methods*
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Protein Subunits / biosynthesis
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Protein Subunits / genetics
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Quinolines
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RNA, Messenger / analysis
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RNA, Messenger / biosynthesis*
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Sensitivity and Specificity
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Spleen / cytology
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T-Lymphocytes / drug effects
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T-Lymphocytes / metabolism
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Tetradecanoylphorbol Acetate / pharmacology
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Transforming Growth Factor beta / biosynthesis
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Transforming Growth Factor beta / genetics
Substances
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Actins
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Benzothiazoles
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Cytokines
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Diamines
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Fluorescent Dyes
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Indicators and Reagents
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Interleukin-12 Subunit p40
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Interleukin-2
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Organic Chemicals
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Protein Subunits
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Quinolines
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RNA, Messenger
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Transforming Growth Factor beta
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SYBR Green I
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Interleukin-12
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Ionomycin
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Interferon-gamma
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Tetradecanoylphorbol Acetate