Real-Time RT-PCR Assays for miRNA Expression Profiling were developed using a novel bioinformatics algorithm for miRNA primer design by incorporating many design filters to improve miRNA assay specificity, sensitivity and homogeneity.
- Multiplexed RT reaction to dramatically increase assay efficiency
- Much more sensitive than TaqMan assays at detecting miRNA expression
- Three specific primers for each miRNA to guarantee assay specificity
- Reliable detection of single nucleotide variations in the miRNA sequence
- High reproducibility when applied to challenging clinical specimens
The overall experimental strategy using these new miRNA assays is summarized in the figure above. A multiplexed RT reaction is performed using a pool of RT primers initially. These RT primers anneal specifically to the target miRNAs, leading to a pool of mixed cDNA products from the miRNAs of interest. By performing a multiplexed RT reaction, the overall experimental cost is significantly reduced since only one RT reaction is needed for each RNA sample. After the RT reaction, separate real-time PCR assays are performed to quantify the expression of individual miRNAs. Each PCR is performed with two primers that are uniquely associated with the miRNA of interest, one with miRNA-specific sequence and the other from the unique tag sequence in the RT primer. Thus, these primers are not likely to cross-react to primers from other miRNA assays even though multiplexed RT reactions are performed. The design algorithm avoids primer cross-reactivity, potential primer dimer and secondary structure formation to ensure uniform PCR assay properties, ensuring all miRNA assays can be performed together under the same experimental condition.
Nawgen miRNA qPCR assays have been extensively validated and functionally tested experimentally in both humans and mice. Our assays can sensitively and reliably quantify just a few miRNA copies and detect a single nucleotide variation in the miRNA sequence. These miRNA qPCR assays have proven to have high reproducibility when applied to challenging clinical specimens in many published studies.