Isolation of RNA from peripheral blood cells: A validation study for molecular diagnostics by microarray and kinetic RT-PCR assays - Application in aerospace medicine

Gene expression studies in clinical diagnostic settings involve a large number of samples collected at different time points requiring effective methods for collection, transportation, storage, and isolation of RNA to maintain the integrity of expression profiles. Human whole blood is a vital source of RNA for analysis of environmental exposure since blood constituents maintain homeostasis, effect immunity or inflammation, participate in stress signaling, and mediate cellular communication in vascular associated tissues including those of the central nervous system. Isolation strategies using whole blood should recognize the limited quantities of useful RNA that must be protected from the hostile leukocyte ribonucleases, in addition to the abundance of preformed mRNA in reticulocytes, high protein content, and transcriptional activation of cells during sample processing in vitro. This paper presents data showing how the collection, treatment, and storage of collected blood samples can affect subsequent RNA isolation and analysis. It is further demonstrated that total RNA isolated from human whole blood, using a modified and optimized procedure of PAXgene(TM) Blood RNA reagent kits, performed well in cDNA microarray hybridization and kinetic RT-PCR. Preservation of expression patterns was observed for 96% of the mRNAs after 24 h storage at 4 C by hybridization analysis of 100 mRNA targets. There were no detectable changes in expression levels of 2 housekeeping genes, beta-actin and cyclophilin, for up to 10 days storage at 4 C by RT-PCR. This validated protocol was employed for isolation of RNA from blood samples collected in the study of acute ethanol effects on performance and characterization of ethanol inducible biomarkers related to performance impairment. The ultimate goal is to utilize gene expression analysis for aerospace accident investigation and prevention.