ANALYSIS OF CYTOKINE GENE-EXPRESSION IN STIMULATED T-CELLS OF SMALL CHILDREN BY SEMIQUANTITATIVE PCR

ONLY Limited amounts of peripheral blood samples can be obtained from small children. Therefore, a polymerase chain reaction (PCR) aided ana lysis of cytokine gene expression by PBMC or T cells is a valuable too l. We present a combination of procedures to obtain an accurate estima tion of the expression of the cytokines IL-4 and IFN-gamma. This can b e performed on T cells purified from blood samples of up to 5 ml in vo lume from children aged 0-4 years with allergic asthma and atopic derm atitis. This procedure includes multiple sampling of PCR products to d etermine the linear phase of the PCR; inter-experiment correction usin g a helper T-cell clone, expressing both IL-4 and IFN-gamma; interpati ent correction by comparing the expression of a housekeeping gene (HPR T); and finally the development of specific software to analyse densit ometric data obtained by scanning photographs of agarose gels, separat ing PCR products. In this way it is possible to study cytokine gene ex pression from a very small amount of material.