Superior assay sensitivity
The miRCURY LNA™ microRNA PCR System is optimized for microRNA quantitation from as little as 10 pg total RNA, which corresponds to the RNA from just a single cell (Figure 4). The system can reliably detect 10 microRNA copies (Figure 3).

The design of PCR primers for microRNA quantitation presents a challenge due to the short size of the microRNA target. Standard PCR protocols applied to microRNA would require primers to be full length compared to the target microRNA. The risk of this approach is the formation of primer-dimers in the reaction, which would result in high background signal. The incorporation of LNA™ nucleosides makes it possible to make very short and yet microRNA-specific primers without sequence overlap, thereby minimizing the risk of primer-dimer formation. This results in extremely sensitive assays with a wide dynamic range of more than eight orders of magnitudes (Figure 3).

Highly specific assays
A mismatch between the microRNA target and the short primer will greatly affect the Tm of the resulting duplex, thereby also affecting the degree to which the target is amplified. The increase of Tm obtained by each substitution of an LNA™ nucleoside into the PCR primer provides higher binding affinity and higher specificity. The difference in amplification of a perfect matched and a mismatched target is, therefore, increased by the incorporation of LNA™ nucleosides in the PCR primers. This means that even very short LNA™-based primers still maintain high sequence specificity. Hence, the result of using a microRNA-specific primer in the RT step together with this short microRNA-specific LNA™ primer in the real-time step is a system with superior specificity and the ability to distinguish between closely related microRNAs that differ in sequence by only one nucleotide.

Moreover, the increased binding affinity obtained from LNA™-enhanced primers improves, and in some cases even rescues, an otherwise poorly performing DNA-based real-time PCR assay (Figure 2).

In addition, the real-time PCR system offers excellent discrimination between mature and precursor microRNAs.

 

Reproducible results
A simple protocol and validated high-quality microRNA primer sets allow for generation of highly reproducible results when testing assay-to-assay variability and robustness (Fig 5). Comparison of individual runs performed on independent days for 30 microRNA assays demonstrated an average correlation coefficient of R2 = 0.9865 for all assays.

Compatible with all real-time PCR instruments
The miRCURY LNA™ microRNA PCR System works well on all commonly used real-time PCR instruments. Figure 6 demonstrates similar and sensitive amplification of the same microRNA assays when using three of the most commonly applied real-time PCR instruments: Roche Lightcycler 480, ABI Prism 7500, and MJ Research Opticon 2.

Ideal for high-throughput quantitation and validation studies
The incorporation of LNA™ monomers in the primers enables Tm-normalization of all microRNA primer sets. This makes it possible to quantitate multiple microRNAs optimally in the same experiment for high-throughput screening. The miRCURY LNA™ microRNA PCR System complements miRCURY LNA™ arrays making the PCR system ideal for validation of microRNA profiling data.

Positive control assay
A primer mix targeting 5S rRNA is provided with the miRCURY LNA™ SYBR® Green Master Mix. Used in combination with the random hexamer provided in the miRCURY LNA™ First-strand cDNA kit, the 5S rRNA primer mix constitutes a positive control assay that can be used for multiple purposes such as assisting in assay setup and troubleshooting.