Direct RT-qPCR analysis of microRNAs in human plasma (miR-Direct)

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Our goal in this proposal is to develop a novel method for the direct detection of miRNAs in serum and plasma without requiring isolation of total RNA. Distinct expression profiles of microRNAs (miRNAs) have recently been associated with cancer and other diseases, implying that miRNAs could serve as diagnostic biomarkers for these diseases. Effective biomarkers could facilitate early detection of cancer, leading to improved therapeutic outcomes, and aid in monitoring progression and response to therapy. The RT-qPCR assays currently employed to quantify circulating miRNAs are hampered by problems associated with their requirement for isolating total RNA from plasma or serum prior to miRNA quantification. RNA is typically isolated by spin-column purification or phenol extraction and ethanol precipitation. Both methods suffer from inconsistent miRNA recovery and the lack of an internal reference miRNA for data normalization. Moreover, current RNA isolation procedures do not completely remove inhibitors of enzymes used in the subsequent RT and PCR reactions. Therefore, RNA preparations cannot simply be concentrated to improve the detection level of the assays. As a result of these issues, the current RT-qPCR methods are best suited for quantification of high-abundance circulating miRNAs. However, many miRNAs that were identified by deep sequencing occur in blood samples at concentrations too low to be reliably detected by current RT-qPCR methods. Thus, there is a need for a method by which miRNA in plasma or serum samples can be quantifies directly, without prior RNA isolation. Direct miRNA sampling allows data normalization to the sample volume and improves both the accuracy and sensitivity of RT-qPCR assays. However, it requires robust methods of separating miRNAs from lipid/protein complexes. Here, we propose a new method, miR-Direct, that provides direct detection of miRNAs from plasma samples and allows use of larger sample volumes through the capture and enrichment of miRNAs of interest before detection. In preliminary studies we have demonstrated that enrichment of plasma miRNAs significantly increases the sensitivity of detection by both the TaqMan(R) assay and SomaGenics' own miR-ID(R) assay. In Phase I, we will develop and optimize methods for liberating miRNAs from plasma complexes and subsequent capture of those miRNAs of interest. We will compare the sensitivity of SomaGenics' miR-ID assay with TaqMan miRNA assay in quantifying the captured miRNAs, and compare both with standard assays performed on purified total RNA from plasma. Then, using ten different plasma samples, we will test the optimized assay on a panel of 10 miRNAs that are associated with various cancers and are present at different abundances in plasma. We expect to demonstrate that miR-Direct can quantify low abundance miRNAs that are not reliably detected by current RT-qPCR assays. In Phase II, we will use the optimized miR-Direct method to validate miRNA biomarker candidates for a specific cancer type and develop a diagnostic test kit for this cancer, to be commerciaized in a partnership with a leading diagnostic provider.