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. 1995 May;4(3):196–204. doi: 10.1155/S0962935195000329

Analysis of cyt0kine gene expression in stimulated T cells of small children by semi-quantitative PCR

H Koning 1,2,✉, M R M Baert 1,2, R de Groot 2, H J Neijens 2, H F J Savelkoul 1
PMCID: PMC2365628  PMID: 18475639

Abstract

Only limited amounts of peripheral blood samples can be obtained from small children. Therefore, a polymerase chain reaction (PCR) aided analysis of cytokine gene expression by PBMC or T cells is a valuable tool. We present a combination of procedures to obtain an accurate estimation of the expression of the cytokines IL-4 and IFN-γ. This can be performed on T cells purified from blood samples of up to 5 ml in volume from children aged 0–4 years with allergic asthma and atopic dermatitis. This procedure includes multiple sampling of PCR products to determine the linear phase of the PCR; inter-experiment correction using a helper T-cell clone, expressing both IL-4 and IFN-γ; interpatient correction by comparing the expression of a housekeeping gene (HPRT); and finally the development of specific software to analyse densitometric data obtained by scanning photographs of agarose gels, separating PCR products. In this way it is possible to study cytokine gene expression from a very small amount of material.

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Selected References

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  1. Chelly J., Kaplan J. C., Maire P., Gautron S., Kahn A. Transcription of the dystrophin gene in human muscle and non-muscle tissue. Nature. 1988 Jun 30;333(6176):858–860. doi: 10.1038/333858a0. [DOI] [PubMed] [Google Scholar]
  2. Corrigan C. J., Haczku A., Gemou-Engesaeth V., Doi S., Kikuchi Y., Takatsu K., Durham S. R., Kay A. B. CD4 T-lymphocyte activation in asthma is accompanied by increased serum concentrations of interleukin-5. Effect of glucocorticoid therapy. Am Rev Respir Dis. 1993 Mar;147(3):540–547. doi: 10.1164/ajrccm/147.3.540. [DOI] [PubMed] [Google Scholar]
  3. Ehlers S., Smith K. A. Differentiation of T cell lymphokine gene expression: the in vitro acquisition of T cell memory. J Exp Med. 1991 Jan 1;173(1):25–36. doi: 10.1084/jem.173.1.25. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Ercolani L., Florence B., Denaro M., Alexander M. Isolation and complete sequence of a functional human glyceraldehyde-3-phosphate dehydrogenase gene. J Biol Chem. 1988 Oct 25;263(30):15335–15341. [PubMed] [Google Scholar]
  5. Gauchat J. F., Lebman D. A., Coffman R. L., Gascan H., de Vries J. E. Structure and expression of germline epsilon transcripts in human B cells induced by interleukin 4 to switch to IgE production. J Exp Med. 1990 Aug 1;172(2):463–473. doi: 10.1084/jem.172.2.463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gilliland G., Perrin S., Blanchard K., Bunn H. F. Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2725–2729. doi: 10.1073/pnas.87.7.2725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Holt P. G., Clough J. B., Holt B. J., Baron-Hay M. J., Rose A. H., Robinson B. W., Thomas W. R. Genetic 'risk' for atopy is associated with delayed postnatal maturation of T-cell competence. Clin Exp Allergy. 1992 Dec;22(12):1093–1099. doi: 10.1111/j.1365-2222.1992.tb00135.x. [DOI] [PubMed] [Google Scholar]
  8. Jolly D. J., Okayama H., Berg P., Esty A. C., Filpula D., Bohlen P., Johnson G. G., Shively J. E., Hunkapillar T., Friedmann T. Isolation and characterization of a full-length expressible cDNA for human hypoxanthine phosphoribosyl transferase. Proc Natl Acad Sci U S A. 1983 Jan;80(2):477–481. doi: 10.1073/pnas.80.2.477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Krug M. S., Berger S. L. First-strand cDNA synthesis primed with oligo(dT). Methods Enzymol. 1987;152:316–325. doi: 10.1016/0076-6879(87)52036-5. [DOI] [PubMed] [Google Scholar]
  10. Leung D. Y. Role of IgE in atopic dermatitis. Curr Opin Immunol. 1993 Dec;5(6):956–962. doi: 10.1016/0952-7915(93)90112-6. [DOI] [PubMed] [Google Scholar]
  11. Mullis K. B. The polymerase chain reaction in an anemic mode: how to avoid cold oligodeoxyribonuclear fusion. PCR Methods Appl. 1991 Aug;1(1):1–4. doi: 10.1101/gr.1.1.1. [DOI] [PubMed] [Google Scholar]
  12. Nakajima-Iijima S., Hamada H., Reddy P., Kakunaga T. Molecular structure of the human cytoplasmic beta-actin gene: interspecies homology of sequences in the introns. Proc Natl Acad Sci U S A. 1985 Sep;82(18):6133–6137. doi: 10.1073/pnas.82.18.6133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Paliard X., de Waal Malefijt R., Yssel H., Blanchard D., Chrétien I., Abrams J., de Vries J., Spits H. Simultaneous production of IL-2, IL-4, and IFN-gamma by activated human CD4+ and CD8+ T cell clones. J Immunol. 1988 Aug 1;141(3):849–855. [PubMed] [Google Scholar]
  14. Patterson S. D., Latter G. I. Evaluation of storage phosphor imaging for quantitative analysis of 2-D gels using the Quest II system. Biotechniques. 1993 Dec;15(6):1076–1083. [PubMed] [Google Scholar]
  15. Punnonen J., Aversa G., Cocks B. G., McKenzie A. N., Menon S., Zurawski G., de Waal Malefyt R., de Vries J. E. Interleukin 13 induces interleukin 4-independent IgG4 and IgE synthesis and CD23 expression by human B cells. Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3730–3734. doi: 10.1073/pnas.90.8.3730. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Pène J. Heterogeneity of atopy demonstrated by differences in cytokine release by peripheral blood T cells. Ann Allergy. 1993 Sep;71(3):322–326. [PubMed] [Google Scholar]
  17. Pène J., Rousset F., Brière F., Chrétien I., Bonnefoy J. Y., Spits H., Yokota T., Arai N., Arai K., Banchereau J. IgE production by normal human lymphocytes is induced by interleukin 4 and suppressed by interferons gamma and alpha and prostaglandin E2. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6880–6884. doi: 10.1073/pnas.85.18.6880. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Tan S. S., Weis J. H. Development of a sensitive reverse transcriptase PCR assay, RT-RPCR, utilizing rapid cycle times. PCR Methods Appl. 1992 Nov;2(2):137–143. doi: 10.1101/gr.2.2.137. [DOI] [PubMed] [Google Scholar]
  19. Wang A. M., Doyle M. V., Mark D. F. Quantitation of mRNA by the polymerase chain reaction. Proc Natl Acad Sci U S A. 1989 Dec;86(24):9717–9721. doi: 10.1073/pnas.86.24.9717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Wierenga E. A., Snoek M., de Groot C., Chrétien I., Bos J. D., Jansen H. M., Kapsenberg M. L. Evidence for compartmentalization of functional subsets of CD2+ T lymphocytes in atopic patients. J Immunol. 1990 Jun 15;144(12):4651–4656. [PubMed] [Google Scholar]
  21. Yssel H., De Vries J. E., Koken M., Van Blitterswijk W., Spits H. Serum-free medium for generation and propagation of functional human cytotoxic and helper T cell clones. J Immunol Methods. 1984 Aug 3;72(1):219–227. doi: 10.1016/0022-1759(84)90450-2. [DOI] [PubMed] [Google Scholar]
  22. de Vries J. E., Gauchat J. F., Aversa G. G., Punnonen J., Gascan H., Yssel H. Regulation of IgE synthesis by cytokines. Curr Opin Immunol. 1991 Dec;3(6):851–858. doi: 10.1016/s0952-7915(05)80003-2. [DOI] [PubMed] [Google Scholar]

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