Gene expression profiling of cells of the immune system isolated ex vivo is a unique tool to assess gene expression in vivo. Exemplified for CD4+CD45RO+ effector/memory T helper (TE/M) lymphocytes of human peripheral blood, we have analyzed the influence of different isolation procedures and storage conditions on the gene expression profile.
Procedures included the use of erythrocyte lysis buffer, ficoll gradient centrifugation, direct and indirect enrichment by magnetic bead assay as well as the addition of the transcriptional blocker ActinomycinD. Additionally, T cells were intentionally activated, using ionomycin and phorbol myristate acetate. Samples were either processed directly or kept at 4°C or room temperature for 2 or 6 hours before TE/M cells were isolated for extraction of total RNA.
All isolation effects were then evaluated by microarray analysis. In consequence identifying patterns of artificially induced gene expression could be described and a method for the generation of unbiased transcriptomes could be established. These results are now implemented in our on going search for biomarkers of diagnostic and therapeutic value in chronic inflammatory diseases.