Elsevier

Methods

Volume 25, Issue 4, December 2001, Pages 386-401
Methods

Regular Article
An Overview of Real-Time Quantitative PCR: Applications to Quantify Cytokine Gene Expression

https://doi.org/10.1006/meth.2001.1261Get rights and content

Abstract

The analysis of cytokine profiles helps to clarify functional properties of immune cells, both for research and for clinical diagnosis. The real-time reverse transcription polymerase chain reaction (RT-PCR) is becoming widely used to quantify cytokines from cells, body fluids, tissues, or tissue biopsies. Being a very powerful and sensitive method it can be used to quantify mRNA expression levels of cytokines, which are often very low in the tissues under investigation. The method allows for the direct detection of PCR product during the exponential phase of the reaction, combining amplification and detection in one single step. In this review we discuss the principle of real-time RT-PCR, the different methodologies and chemistries available, the assets, and some of the pitfalls. With the TaqMan chemistry and the 7700 Sequence Detection System (Applied Biosystems), validation for a large panel of murine and human cytokines and other factors playing a role in the immune system is discussed in detail. In summary, the real-time RT-PCR technique is very accurate and sensitive, allows a high throughput, and can be performed on very small samples; therefore it is the method of choice for quantification of cytokine profiles in immune cells or inflamed tissues.

References (91)

  • J Rengarajan et al.

    Immunol. Today

    (2000)
  • M Baggiolini et al.

    Immunol. Today

    (2000)
  • Z Dai et al.

    Curr. Opin. Immunol.

    (1999)
  • T Wang et al.

    Anal. Biochem.

    (1999)
  • R.A Montgomery et al.

    Cytokine

    (1997)
  • N.M Zhou et al.

    Cytokine

    (1997)
  • A O'Garra et al.

    Curr. Opin. Immunol.

    (1992)
  • L.M Kuschnaroff et al.

    J. Neuroimmunol.

    (1999)
  • C Härtel et al.

    J. Immunol. Methods

    (2001)
  • V Blaschke et al.

    J. Immunol. Methods

    (2000)
  • M.B Oleksiewicz et al.

    J. Virol. Methods

    (2001)
  • P.O Van Trappen et al.

    Lancet

    (2001)
  • P.S Bernard et al.

    Am. J. Pathol.

    (1998)
  • G Schalasta et al.

    J. Clin. Virol.

    (2000)
  • A Moody et al.

    J. Virol. Methods

    (2000)
  • K.J Livak et al.

    Methods

    (2001)
  • H Zhong et al.

    Biochem. Biophys. Res. Commun.

    (1999)
  • C.J Gerard et al.

    Mol. Diagn.

    (2000)
  • A Thomson

    The Cytokine Handbook

    (1998)
  • E.J Corwin

    Biol. Res. Nurs. Pt. I

    (2000)
  • L.H Glimcher et al.

    Genes Dev.

    (2000)
  • A Rabinovitch

    Diabetes Metab. Rev.

    (1998)
  • K Mullis et al.

    Cold Spring Harb. Symp. Quant. Biol.

    (1986)
  • L.M Kuschnaroff et al.

    Scand. J. Immunol.

    (1997)
  • C.J Fox et al.

    J. Immunol.

    (1997)
  • G Gilliland et al.

    Proc. Natl. Acad. Sci. USA

    (1990)
  • S Barth et al.

    Inflamm. Res.

    (2000)
  • L Raeymarkers

    Anal. Biochem.

    (1993)
  • M.S Hanson et al.

    J. Immunol.

    (1996)
  • P.M Holland et al.

    Proc. Natl. Acad. Sci. USA

    (1991)
  • L Stryer

    Annu. Rev. Biochem.

    (1978)
  • R.A Cardullo et al.

    Proc. Natl. Acad. Sci. USA

    (1998)
  • C.A Heid et al.

    Genome Res.

    (1996)
  • U.E.M Gibson et al.

    Genome Res.

    (1996)
  • Overbergh, L., Valckx, D., Waer, M., and Mathieu, C. (1999) Cytokine11,...
  • K.M Casteels et al.

    Transplantation

    (1998)
  • C.A Gysemans et al.

    Diabetes

    (2000)
  • K.M Casteels et al.

    Endocrinology

    (1998)
  • K.M Casteels et al.

    Clin. Exp. Immunol.

    (1998)
  • C.A Gysemans et al.

    Diabetologia

    (2001)
  • M.-C Chen et al.

    Diabetologia

    (2001)
  • H Sefrioui et al.

    Transplantation

    (1999)
  • L Overbergh et al.

    Diabetes

    (2000)
  • A.D Billiau et al.

    Transplantation

    (2001)
  • B Decallonne et al.

    Diabetologia

    (2000)
  • Cited by (1147)

    View all citing articles on Scopus
    1

    Both authors contributed equally to this review.

    2

    To whom correspondence should be addressed. Fax: 00 32 16 345934. E-mail: [email protected]

    View full text