MicroRNA profiling using a rapid, highly sensitive qPCR panel

Simon Baker1, Lihan Zhou2, Florent Chang Pi Hin1, Ruiyang Zou2, Heng-Phon Too2
1Bioline Reagents Ltd, United Kingdom; 
2MIRXES Pte. Ltd, Singapore

Recent studies have shown that microRNAs are released from tissues and cells into circulation where their expression correlates with specific disease conditions. These microRNAs are protein bound or encapsulated in vesicles and remain stable in biofluids. Such attributes made circulating microRNAs promising non-invasive candidate biomarkers for the early detection of disease, prognosis or treatment selection. However, significant technological challenges remain. These include data inconsistencies across studies which has hampered the development of accurate and robust miRNA-based tests. Efficient identification of miRNA biomarkers requires a well-designed study and an integrated workflow with consistent sample extraction, robust RT-qPCR quantification, and stringent statistical analysis. In addition, control measures are essential to monitor and normalize technical or biological variations that may obstruct the interpretation of data. We have developed an enabling platform based on a proprietary design concept and validated the resulting qPCR assays against both clinical samples and synthetic RNA constructs. We have detected extremely low levels of circulating microRNAs with high sensitivity and specificity. This new platform has been used for the routine detection of over 400 microRNAs, using low input material (e.g. 200 microliters of serum and plasma) with a dynamic range span over eight orders of magnitude. The total workflow variation was less than 0.5 Ct. The platform has been validated independently by molecular diagnostic and pharmaceutical laboratories and showed superior performance to existing commercial assays. This highly reproducible, sensitive platform has been developed to produce complete systems for miRNA profiling of cancer samples, biofluids and stem cells.

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