Locked nucleic acids (LNA) are a class of high-affinity RNA analogs in which the ribose ring is "locked" into the ideal conformation for Watson-Crick binding. These oligonucleotides have substantially increased affinity for their complementary strand, when compared with traditional DNA or RNA oligonucleotides. This results in unprecedented sensitivity and specificity and makes LNA-enhanced oligos excellent tools for detecting and differentiating small or highly similar DNA or RNA targets in many research applications. Discover more about LNA in our Learning Hub.

Unique systems developed specifically for miRNA profiling

Both the miRCURY LNA miRNA PCR System, which uses SYBR Green for detection, and the new probe-based miRCURY LNA miRNA Probe PCR System offer the best available combination of performance and ease-of-use on the market through a unique combination of features, but differ in detection strategy.

• Both systems use one cDNA reaction for all miRNAs – A single universal first-strand cDNA synthesis reaction is used as the template, regardless of the number of miRNAs being profiled. This saves precious sample, reduces technical variation, minimizes pipetting and saves time in the laboratory.
• The miRCURY LNA miRNA PCR System uses two LNA-enhanced, miRNA-specific qPCR primers and SYBR Green for the signal detection (see Schematic outline of the miRCURY LNA miRNA PCR System). The miRCURY LNA miRNA Probe PCR System uses an LNA-enhanced, miRNA-specific forward primer and an LNA-enhanced, miRNA-specific hydrolysis probe (see miRCURY LNA miRNA Probe PCR System at a glance). LNA-enhancement ensures that the primers of both systems and the probe of the latter bind with high affinity. Primers and probe are also shorter than standard PCR primers, so they can be designed to be fully miRNA-specific without overlapping. The result is unrivalled sensitivity and specificity and extremely low background.

Universal RT combined with LNA-enhanced and Tm normalized primers and probe enables accurate and reliable quantification of individual miRNAs from as little as 1 pg total RNA (see Accurate quantitation even with low starting material). In comparison to other miRNA real-time PCR systems that use either stem-loop or standard DNA primers, the LNA-enhanced primers offer significantly increased sensitivity, especially for AU-rich miRNAs.

Exceptional sensitivity and extremely low background enable accurate quantification of very low levels of miRNA without the need for pre-amplification. This makes the two miRCURY LNA miRNA System, both SYBR Green and probe-based, suitable for all sample types, and especially samples with low RNA content, such as biofluids like serum and plasma.

All miRNA assays for the two systems have been optimized for maximum sensitivity and thoroughly validated either by wet-lab testing or in silico. In wet-lab validation, over 95% of miRCURY LNA miRNA PCR Assays detected 10 miRNA copies or less in the PCR reaction. In general, both systems have high assay efficiency (see High efficiency of the two miRCURY LNA miRNA PCR Systems) and the miRCURY LNA miRNA Probe PCR System shows improved efficiency compared to other systems (see Improved efficiency compared to alternative solution). The primer sets have also been validated for specific amplification of the target and for minimal background signal.

The incorporation of LNA in the qPCR primers facilitates the design of assays that can distinguish between miRNA sequences that differ by a single nucleotide. This makes these two systems truly specific miRNA qPCR platforms that give virtually no false-positive signals (see Specificity results from the miRQC Study and Discriminatory power down a single nucleotide difference). In addition, the assays can discriminate between mature miRNA sequences and precursor miRNA.

Save time and effort in the laboratory with the < 3-hour, easy-to-follow protocol of each system that minimizes pipetting. By using the same cDNA as the template for all subsequent PCR reactions, the procedure is greatly simplified compared to systems that require miRNA-specific cDNA synthesis. With fewer pipetting steps, technical variation is also reduced. As a result, it is possible to achieve extremely high reproducibility from day-to-day (see Excellent day-to-day reproducibility), site-to-site and lot-to-lot (see High lot-to-lot consistency). Furthermore, the same cDNA template can be used for all assay and panel formats of a system, making it easy to adjust throughput and format as needed and use samples efficiently.

Tailor your experimental setup to your specific needs using individual assays or fully flexible custom panels. Using the same cDNA reaction, shift seamlessly between individual primers, pre-designed miRNome and Focus panels or custom panels.

Implementing each system begins with the use of a universal reverse transcription kit (1) that contains the chemistry for both SYBR Green and probe-based detection. Then, dedicated mastermixes for each system (3) are combined with your choice of pre-designed or self-designed assays or panels (4). Optionally, RNA spike-ins can be added to samples as a quality control for RNA isolation and cDNA synthesis (2).