miRNA Quantification Using LNA qPCR

These unique qPCR systems give you the freedom to choose SYBR Green or probe-based miRNA detection within the context of technologies that merge performance and ease-of-use: the speed of a universal reverse transcription reaction and the sensitivity and specificity of LNA-enhanced qPCR primers. Designed for high-performance miRNA profiling, the easy workflow delivers fast, accurate results and is well-suited for all sample types – especially those with low RNA content.

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.
Schematic outline of the miRCURY LNA miRNA PCR System. A poly(A) tail is added to the mature miRNA template (step 1A). cDNA is synthesized using a Poly(T) primer with a 3’ degenerate anchor and a 5’ universal tag (step 1B). The cDNA template is then amplified using two miRNA-specific and LNA-enhanced forward and reverse primers (step 2A). SYBR Green is used for detection (step 2B).
miRCURY LNA miRNA Probe PCR System at a glance. The miRCURY LNA miRNA Probe PCR System uses a single cDNA synthesis reaction for all amplifications, reducing pipetting and saving time and sample. A forward PCR primer and hydrolysis probe that are LNA-enhanced and miRNA-specific deliver highly sensitive miRNA quantification and specificity down to a single-nucleotide sequence difference. The fast and easy workflow takes 2 hours with minimal hands-on time.
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.
Accurate quantitation even with low starting material. Designed with LNA‐enhanced, miRNA-specific reverse PCR primers and hydrolysis probes, the miRCURY LNA miRNA Probe PCR System delivers highly sensitive results enabling the miRNA detection from just 1 pg starting material. This robust miRNA quantification system shows high performance across a dynamic range that spans most clinically relevant sample types.
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.
High efficiency of the two miRCURY LNA miRNA PCR Systems. The two systems use different detections methods – one uses SYBR Green and the other uses an LNA-enhanced probe. Both systems demonstrate exceptional linearity and efficiency with the SYBR Green detection method showing slightly lower Cq values.
High efficiency of the two miRCURY LNA miRNA PCR Systems. Four miRNA targets were diluted 10‐fold and the resulting concentrations were quantified with the miRCURY LNA miRNA Probe PCR System or with a solution from Supplier T. Calculated relative expression of each reaction is compared in the graph to the expected fold change. Quantification with miRCURY LNA miRNA Probe PCR System is more consistent with the expected than outcomes of the Supplier T product for nearly every target tested.
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.
Specificity results from the miRQC Study. From Mestdagh et. al., Nature Methods 2014.
Discriminatory power down a single nucleotide difference. The LNA‐enhanced forward primer and probe of the miRCURY LNA miRNA Probe PCR System are short, so both are designed to be target-specific and to not overlap upon binding. This boosts assay specificity to distinguish closely related miRNAs that differ by a single nucleotide.
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.
Excellent day-to-day reproducibility. Different RT reactions using 40 ng heart and liver total RNA were profiled on the miRCURY LNA miRNome PCR Human PCR Panel I and II on different days. The correlation between raw Cq values from all miRNAs with signals below 35 Cq values is shown (total of 297 data points).
High lot-to-lot consistency. Different RT reactions using 40 ng heart and liver total RNA were profiled on the miRCURY LNA miRNome PCR Human PCR Panel I and II on different days. The correlation between raw Cq values from all miRNAs with signals below 35 Cq values is shown (total of 297 data points).
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).
  Detection system SYBR Green LNA-enhanced hydrolysis probe
1 Reverse Transcription miRCURY LNA RT Kit miRCURY LNA RT Kit
2 Optional spike-in RNA Spike-In Kit, For RT RNA Spike-In Kit, For RT
3 Mastermix for qPCR miRCURY LNA SYBR Green PCR Kits miRCURY LNA Probe PCR Kits
4 Assay or panel miRCURY LNA miRNA PCR Assays
• miRCURY LNA miRNA Custom PCR Assays
• miRCURY LNA miRNA Focus PCR Panels
• miRCURY LNA miRNA Custom PCR Panels
• miRCURY LNA miRNA miRNome PCR Panels

• miRCURY LNA miRNA Probe PCR Assays
• miRCURY LNA miRNA Custom Probe PCR Assays

Coming soon:
• miRCURY LNA miRNA Focus Probe PCR Panels
• miRCURY LNA miRNA Custom Probe PCR Panels
• miRCURY LNA miRNA miRNome Probe PCR Panels

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