Gene expression changes in blood after phlebotomy: implications for gene expression profiling.

Rapidly developing technologies such as quantitative real-time reverse transcriptase–polymerase chain reaction (RT-PCR) and microarrays enable characterization and quantification of nucleic acids in clinical samples. Identification of DNA from pathogens such as bacteria or viruses is already widely used in molecular diagnostics. 1 The use of RNA as a diagnostic tool is currently being investigated. Gene expression profiling of tumor samples using microarray technology has become the prototypic application for this new type of molecular diagnostics. Cluster analysis of large-scale microarrays revealed that characterization of only a few genes is sufficient to distinguish different tumors. 2 However, levels of gene expression may change between sample acquisition and the beginning of analysis, and these changes are not well understood. Blood is frequently used for diagnostic sampling and processing. However, differences in blood collection and preparation techniques may cause changes in gene expression levels ex vivo and consequently affect the resulting mRNA expression profiles. For example, several investigators have shown that anticoagulants affect leukocyte viability and cause ex vivo changes in cytokine production. 3,4 The extent to which events after blood collection influence gene expression is not known. To our knowledge, changes in gene expression between phlebotomy and the beginning of analysis have not been studied. We evaluated the effect of ex vivo incubation of blood on the gene expression status of human peripheral blood. After informed consent, blood was obtained from healthy donors. Whole blood collected in EDTA (ethyl-enediaminetetraacetic acid) tubes was stored under sterile conditions at room temperature for different periods of time. Gene expression was monitored by quantitative real-time RT-PCR using TaqMan assays on an ABI PRISM 7700 (Applied Biosystems, Weiterstadt, Germany). Primer and probe sequences are available upon request. mRNA quantity was calculated using the ⌬⌬C T method (PE Applied Biosystems User Bulletin #2; ABI PRISM 7700 Sequence Detection System, 1997). We studied the changes in transcript levels of proinflamma-tory genes known to be sensitive to extracellular stimuli in whole blood after phlebotomy. Figure 1 shows the changes in levels of IL-1␤, IL-6, and TNF␣ transcripts up to 7 days. Levels of IL-6 and TNF␣ transcripts increased over the entire observation period, reaching maximum levels of about 20-fold higher than levels measured immediately after blood collection. IL-1␤ transcripts showed a different pattern. After 6 hours a decrease in expression was observed, followed by an increase after 1 and 3 days. Seven days after phlebotomy, IL-1␤ mRNA levels again decreased …