Stabilization of Gene Expression Profiles in Blood after Phlebotomy

Abstract

Emerging technologies such as quantitative, real-time reverse transcription-PCR (RT-PCR) and microarrays enable the accurate quantification of ribonucleic acids in clinical samples. Gene expression profiling of tumor samples with use of microarray technology has become the prototypic application of this new type of molecular diagnostics. DNA microarray analysis of different closely related tumor types revealed that this technology is able to distinguish different tumors (1). The concentrations of specific mRNAs, however, may change between specimen acquisition and the beginning of analysis, and these changes are not well understood. When blood is studied, specimen collection and sample preparation techniques by themselves may produce changes in gene expression ex vivo, e.g., it has been shown that anticoagulants cause ex vivo changes in cytokine production (2). If changes in gene expression occur after phlebotomy, the valid interpretation of basal mRNA expression data as sensitive markers in clinical studies requires the standardization of conditions for assaying patient blood samples. The current study was undertaken to analyze changes in gene expression in human peripheral blood stored ex vivo. We studied ex vivo changes in expression of several genes by use of quantitative, real-time RT-PCR and evaluated procedures for standardized blood sampling for molecular diagnostics. After receiving informed consent, we obtained blood from healthy donors (23–60 years of age). Whole blood was collected with use of the VACUTAINER® Safety-LokTM Blood Collection Set and either PAXgeneTM Blood RNA Tubes (2.5 mL draw volume; PreAnalytiX) or EDTA tubes (VACUTAINER PLUSTM 6-mL dipotassium EDTA tubes). This investigation was performed according to the International Declarations of Helsinki and Tokyo. After phlebotomy, PAXgene Blood RNA Tubes were stored at room temperature until processing. Alternatively, immediately after phlebotomy, 1 mL of water and 6 mL of Trizol LS reagent (Invitrogen) were added to 1 mL of whole …