Preparation of Circular Templates by T4 RNA Ligase 2 for Rolling Circle Amplification of Target microRNAs with High Specificity and Sensitivity
Rolling circle amplification (RCA) is a robust bioanalytical method invented based on the principle of viral DNA reproduction. Since RCA offers many technical advantages of convenient operation, cost effective experimental setup, rapid result delivery, etc. it has been widely used for analysis and detection of DNAs, RNAs, and proteins. It was recently shown that the RCA based methods are suitable for analysis of micro RNAs (miRNAs) To optimize the RCA based miRNA analysis, we evaluated ligation efficiencies of T4 RNA ligase 2 for different DNA/RNA heteroduplexes using a molecular beacon based assay. Then, we designed padlock probes, each being specific to particular miRNA, and examined their ability to differentiate between members of the let 7 family of miRNAs having high sequence homology. It was found that each padlock probe could accurately and unambiguously identify the target miRNA at concentrations as low as 2.5 fmol. Results from this study are important for the development of RCA based diagnostics of miRNA biomarkers in clinical and environmental specimens.