Bead array-based microRNA expression profiling of peripheral blood and the impact of different RNA isolation approaches

Abstract

Blood-based mRNA expression profiling has already become an important issue in clinical applications. More recently, the characterization of the small RNA transcriptome offers additional avenues for diagnostic approaches. However, when applying miRNA expression profiling in routine clinical settings, the method of RNA preservation and the manner of RNA extraction as well as the reliability of the miRNA profiling procedure have to be carefully considered. Here we evaluate a recently introduced bead array-based technology as a robust method for the generation of blood-based human miRNA expression profiles. Importantly the comparison of different RNA extraction strategies resulted in dissimilar profiles depending on the RNA extraction method as well as on the underlying source. Expression profiles obtained from peripheral mononuclear cells (PBMCs) substantially differed from those of whole blood samples, whereby both sources per se yielded reproducible and reliable results. Expression profiles were also distinct when using either fresh or frozen PBMCs. Moreover RNA size fractioning resulted in discriminative miRNA expression profiles compared with total RNA based profiles. This study outlines important steps toward the establishment of a robust strategy for blood-based miRNA profiling and provides a reliable strategy for its implementation in routine handling for diagnostic purposes. Copyright © American Society for Investigative Pathology and the Association for Molecular Pathology.