miRNA Quantification Using LNA qPCR

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Discover the miRCURY LNA miRNA PCR System
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This unique miRNA qPCR system offers the best combination of performance and ease-of-use: the speed of a universal reverse transcription reaction, together with the sensitivity and specificity of LNA-enhanced qPCR primers. Designed for high-performance miRNA profiling, the fast and easy workflow delivers accurate results in just 3 hours and is well-suited for all sample types – especially those with low RNA content.
A unique system developed specifically for miRNA profiling
Unmatched sensitivity
Fully validated and optimized
Truly specific – no false positives
Fast, easy and reproducible
Flexible qPCR system
A unique system developed specifically for miRNA profiling
The miRCURY LNA miRNA PCR System offers the best available combination of performance and ease-of-use on the market, because it unites two important features (see figure Schematic outline of the miRCURY LNA miRNA PCR System):
  • One cDNA reaction for all miRNAs – One 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, reduces pipetting and saves time in the laboratory.
  • Two LNA-enhanced miRNA qPCR primers – Both qPCR primers are miRNA-specific and optimized with LNA. LNA primers bind with high affinity and are shorter than standard PCR primers, so both primers can fit on the miRNA without overlapping. The result is unrivalled sensitivity and specificity and extremely low background.

Unmatched sensitivity
Universal RT combined with LNA-enhanced and Tm normalized primers enables accurate and reliable quantification of individual miRNAs, from as little as 1 pg total RNA (see figure Accurate quantitation from down to 1 pg total RNA 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 miRCURY LNA miRNA PCR System suitable for all sample types, and especially samples with low RNA content, such as biofluids like serum and plasma.

Fully validated and optimized
All miRCURY LNA RT miRNA PCR primer sets have been optimized for maximum sensitivity and thoroughly validated either by wet-lab testing or in silico. In wet-lab validation, over 95% of assays detect 10 miRNA copies or less in the PCR reaction (see figure Serial dilution of hsa-miR-181a). The primer sets have also been validated for specific amplification of the target and for minimal background signal.

Truly specific – no false positives
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 it a truly specific miRNA qPCR platform that gives no false-positive signals (see figure Specificity results from the miRQC Study). In addition, the assays can discriminate between mature miRNA sequences and precursor miRNA.

Fast, easy and reproducible
Save time and effort in the laboratory with the 3-hour, easy-to-follow protocol 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 figure Excellent day-to-day reproducibility) and even site-to-site.

Flexible qPCR system
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.
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For extremely sensitive and specific miRNA quantification using LNA-optimized miRNA PCR
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For miRNA quantification using LNA-optimized miRNA PCR primer sets
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For exceptionally sensitive and specific miRNA profiling using LNA-enhanced miRNA qPCR
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Design your own custom-formatted 96- and 384-well qPCR plates for the miRCURY LNA miRNA PCR System
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For exceptionally sensitive and specific miRNA profiling using LNA-enhanced miRNA qPCR
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For setup and optimization of miRNA quantification experiments
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For optimal first-strand cDNA synthesis with the SYBR-based or probe-based miRCURY LNA miRNA PCR Systems
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For high-performance PCR with the miRCURY LNA miRNA PCR System
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Schematic outline of the miRCURY LNA miRNA PCR System.
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).
Accurate quantitation from down to 1 pg total RNA starting material.
Accurate quantitation from down to 1 pg total RNA starting material.
Data from serial dilutions of human AM6000 total RNA are shown. All miRNA assays exhibit linear readout with correlation coefficients R2 > 0.99.
Serial dilution of hsa-miR-181a.
Serial dilution of hsa-miR-181a.
A 10-fold serial dilution (ranging from 1 x 108 to 10 copies) of cDNA template made from synthetic hsa-miR-181a was used for real-time PCR amplification. The standard curve shows excellent linear correlation (R2 = 0.998) between the decreasing cycle numbers and the logarithm of the miRNA copy number. The assay can detect as few as 10 miRNA copies in the PCR reaction and has a dynamic range of 8 logs. Efficiency calculated from the standard curve = 1.94. The dilution series was performed in a background of MS2 bacteriophage total RNA.
Excellent day-to-day reproducibility.
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).
Specificity results from the miRQC Study: Mestdagh et. al., Nature Methods 2014.
Specificity results from the miRQC Study.
From Mestdagh et. al., Nature Methods 2014.