Ditte Andreasen, Niels Tolstrup, Jacob Ulrik Fogh, Søren Jensby Nielsen, Kim Bundvig Barken, Adam Baker, Peter Mouritzen
microRNAs (miRNAs) constitute a recently discovered class of small RNAs (typically 21-23 nt) that function as post-transcriptional regulators of gene expression. Current estimates indicate that more than one third of the cellular transcriptome is regulated by miRNAs, and miRNAs have been proposed to be master regulators of cellular state. Indeed, changes in miRNA expression patterns have been associated with disease states, including a diverse array of human cancers. Furthermore, the high stability of miRNA in common clinical source materials (e.g. FFPE blocks, plasma, serum, urine, saliva, etc.) and the ability of miRNA expression profiles to accurately classify disease states have positioned miRNA quantification as a promising new tool for a wide range of diagnostic applications. To facilitate discovery and clinical transfer of miRNA-based diagnostic markers, we developed the miRNome-wide LNA™-based miRCURY LNA™ Universal RT microRNA PCR platform with unparalleled sensitivity and robustness. Using a specialized design algorithm (available as a web-tool for custom design of RNA species < 30 nt), we have designed assays for human and rodent miRNAs. The platform uses only 40 ng total RNA in just a single RT reaction to profile >700 human miRNAs in two predefined 384 well plates and thus allows high-throughput profiling of miRNAs from important clinical sources without the need for pre-amplification. Following screening, it is possible to choose a sub-set of miRNA assays for validation to be formatted in 96- or 384-well plates using our Pick&Mix platform. The high sensitivity of the assays makes it possible to do high quality microRNA expression profiling in samples that contain very little total RNA, such as sections of formalin fixed paraffin-embedded samples (FFPE) and blood serum and plasma. Results will be presented demonstrating the application of the new qPCR method in the profiling of miRNAs from plasma as part of our efforts to develop molecular diagnostic tests for treatment selection, diagnosis and monitoring of cancer. We will also show results demonstrating how selection of a proper set of miRNA biomarkers in a Pick&Mix plate format can give precise classification of disease progression.
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