A new method for multiplex detection of micro-RNAs (miR-ID)

Kazakov, Sergei

Abstract

A new method for multiplex detection of micro-RNAs (miR-ID) ABSRACT MicroRNAs (miRNAs) and other short non-coding RNAs play diverse biological functions, and distinctive miRNA expression patterns are associated with various types of cancer and viral infections. Thus, miRNAs may be considered as potential diagnostic biomarkers as well as potential drug targets. Many different methods for detecting miRNAs and profiling their expression patterns have been developed recently. However, none of these methods is capable of quantifying different miRNAs with equal accuracy and sensitivity, and all suffer from problems such as laborious handling and purification steps, and expensive equipment and reagents. Here we propose a novel method, which we call miR-ID, that allows fast, accurate, sensitive, and highly multiplexable quantification of short target RNAs such as miRNAs through signal (rather than target) amplification. The miR-ID approach is also potentially less complex and expensive than alternative available methods. MiR-ID employs a probe that contains a miRNA-specific capture sequence as well as signal sequences. This probe is expected to efficiently and sequence-specifically capture target miRNAs directly from nucleic acid extracts or cell lysates. The probe-miRNA complex is then processed in such a way that it can be quantified by qPCR. The proposed detection method has no need for chemical or enzymatic labeling of miRNAs, avoids sequence-dependent bias that could be introduced by miRNA ligation and reverse transcription, is compatible with qPCR machines from a variety of vendors, and allows massive multiplexing. In Phase I, we will test several different designs of capture probes and seek the best combination of sensitivity, sequence-specificity, low cost, and workflow time. We will also verify the sensitivity and specificity of the method in a """"""""real-world"""""""" setting by quantifying cellular miRNAs and comparing our results with published data obtained with other methods. In Phase II, we plan to develop a diagnostic assay for prostate cancer detection as well as for monitoring progression of the disease and response to therapy.

Public Health Relevance

Prostate cancer is the second leading cause of cancer deaths among American men. The goal of this application is early diagnosis of this disease based on quantification of a panel of microRNAs that constitutes a promising biomarker. The method of micro-RNA detection has novel features that promise better sensitivity and selectivity than other micro-RNA assays currently available. ? ? ? ?