MicroRNA isolation and analysis from human whole blood

A108

MicroRNAs (miRNAs) represent an exciting new class of small, regulatory biomolecules. Data to date suggests that as many as half of all genes may be regulated by miRNAs. Over the past few years, scientists have demonstrated that miRNAs play important roles in processes as diverse as early development, cell proliferation and differentiation, apoptosis and fat metabolism, and oncogenesis. Indeed, tumor-associated, differentially expressed miRNAs, termed "oncomirs," have been found in more than a half dozen different cancers and the number of known oncomirs is growing rapidly.Blood is a readily accessible tissue that carries biological material from all reaches of the body, and is the preferred clinical sample for many diagnostic tests. Blood-borne cells such as lymphocytes, monocytes, neutrophils, eosinophils, and basophils contain molecular clues of infection, inflammation, and other hallmarks of disease. We hypothesize that miRNA biomarkers exist in blood, and further that these biomarkers will drive the development of future molecular diagnostics assays. However, technologies for isolating and analyzing the global miRNA profile in human whole blood are largely lacking. For example, the most widely used protocol for whole blood RNA stabilization and isolation, the PAXgene™ Blood RNA System, does not provide for the recovery of small RNA. As a result, we developed a novel methodology using PAXgene stabilized blood that enables the isolation of mature, ~21-mer miRNAs. Using microarrays, we then compared the expression levels of >380 miRNAs in blood with the miRNA profile from nearly two dozen other healthy human tissues. Outlier analysis enabled the identification of miRNAs that were relatively differentially expressed in whole blood with respect to the expression profiles from a large set of reference tissues. The implications of these findings are discussed.