The design process
The miRCURY LNA miRNA Custom PCR Assay design tool lets you easily design highly sensitive and specific LNA-enhanced PCR primer sets for any miRNA not available as a predesigned product. The advanced algorithm evaluates approximately 3,000 primer pair designs based on more than 60 different criteria to find LNA optimal primer sets for your miRNA within a few minutes. The tool has been designed for miRNAs but can also be used for other small RNAs 14–27 nucleotides in length.
The design criteria include:
- Optimization of melting temperatures by varying the LNA distribution of the primers to ensure high amplification efficiency and specificity.
- Calculations and adjustments of self-hybridization and cross-hybridization scores for efficient PCR reactions.
- Adjustments to avoid potential primer–dimer formation in the PCR reaction.
- Intelligent positioning of LNA bases in the primers based on our vast knowledge of LNA oligonucleotide design.
- miRBase searches to identify potential miRNAs with high sequence similarity. This ensures that the primer sets are highly specific for the small RNA for which they were designed.
The exceptional sensitivity of miRCURY LNA miRNA PCR Assays is achieved by combining universal reverse transcription with LNA-enhanced and Tm
-normalized primers. This combination enables accurate and reliable quantification of individual miRNAs from as little as 1 pg of total RNA input in the initial first-strand cDNA synthesis (see figure Accurate quantitation from down to 1 pg total RNA starting material
Compared with 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 AT-rich miRNAs (see figure LNA-enhanced primers result in greatly increased sensitivity compared to DNA primers
The low sample requirements also enable miRNA quantitation using total RNA purified from difficult samples such as LCM samples and serum/plasma (see figures Detection of differential expression of miRNAs in LCM specimens from FFPE tissue sections
and Differences in miRNA expression between serum samples
Fully validated and optimized
All wet-lab validated miRCURY LNA miRNA PCR Assays have been optimized to be as sensitive as possible. Over 80% of assays detect at least 5 miRNA copies 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 (see figure Specific and sensitive amplification of miRNA
The only platform with perfect specificity
The miRCURY LNA miRNA PCR Assays are the only miRNA quantification platform with absolute specificity (see the miRQC study published in Nature Methods
) and out-performed another probe-based miRNA qPCR platform in specificity tests (see figure Specificity test with probe-based miRNA qPCR system
). Perfect specificity eliminates false positives and ensures only robust and reliable miRNA signals.
The incorporation of LNA in both the forward and reverse PCR amplification primers makes it possible to design assays that can distinguish between miRNA sequences that differ by only one nucleotide (see figure Single-nucleotide discrimination
). In addition, the assays can discriminate between mature miRNA sequences and precursor miRNA.
Fast, easy and reproducible
The easy-to-follow, 3-hour protocol saves you both time and effort in the laboratory. By using the same RT reaction as the template in all subsequent PCR reactions, the procedure is greatly simplified compared with systems that require miRNA-specific first-strand synthesis. The number of pipetting steps is reduced to a minimum, and technical variation is minimized. This makes it possible to achieve extremely high reproducibility from day-to-day and even site-to-site.
miRCURY LNA miRNA PCR Assays have been optimized for use with the miRCURY LNA RT Kit and the miRCURY LNA SYBR Green PCR Kit. Use of other reagents will affect the quality of the results.