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Ditte Andreasen, Jacob Ulrik Fogh, William Biggs, Jesper Salomon, Adam Baker, Peter Mouritzen Exiqon A/S, Denmark |
Abstract
MicroRNAs (miRNAs) are currently emerging as a new class of biologically important biomarkers. Altered miRNA expression profiles are associated with a number of different diseases including heart disease, neurological disorders and human cancers. This suggests the use of miRNAs as biomarkers for disease diagnosis and prognosis. The study of microRNA expression is complicated by their small size, high sequence similarity between family members and large variability in CG content. In order for biomarkers to be useful in clinical settings, they should be accurately and reliably detected in clinical samples such as formalin fixed paraffin embedded (FFPE) sections and blood serum or plasma. Analysis of microRNA from such samples is difficult due to low RNA yield and poor quality, setting high demands for the sensitivity, robustness and reproducibility of the assays as well as the experimental design and normalization methods. We here present an LNA™ based, highly sensitive real-time PCR method which, without the need for pre-amplification, is able to reliably quantify small amounts of miRNAs, even from difficult clinical samples. Real-time PCR amplification using LNA™ enhanced miRNA specific primers results in exceptional sensitivity, also for AT rich miRNAs .The method relies on a universal reverse transcription (RT) reaction, which with an input of only 40 ng total RNA will generate sufficient cDNA to enable profiling of an entire panel of ~730 miRNAs. Results will be presented demonstrating the application of the new qPCR method in the large scale profiling of miRNAs from both FFPE samples and blood serum/plasma with special focus on strategies for improving the reliability and accuracy of the results.
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