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miRCURY LNA miRNA Custom PCR Assays

For miRNA quantification using LNA-optimized miRNA PCR primer sets
  • Design your own LNA-enhanced miRNA PCR primer sets for any miRNA
  • Sensitive and specific detection and quantification of miRNA
  • Fast and simple two-step protocol takes less than 3 hours

miRCURY LNA miRNA Custom PCR Assays are custom-designed miRNA PCR primer sets that enable extremely sensitive and specific miRNA quantification with the miRCURY LNA miRNA PCR System. Both forward and reverse PCR amplification primers are miRNA-specific and are optimized with LNA technology. Each tube contains sufficient assays for 200 reactions.

The former Exiqon "miRCURY LNA Universal RT microRNA PCR System" has been updated to the "miRCURY LNA miRNA PCR System" with new reagents that ensure the same superior performance:

  • The Universal cDNA Synthesis Kit II has been replaced by the miRCURY LNA RT Kit
  • The ExiLENT SYBR Green Master Mix Kit has been replaced by the miRCURY LNA SYBR Green PCR Kit
  • Most miRCURY LNA miRNA PCR Assays remain unchanged, but 15 validated assays have been redesigned; refer to the Product Resources tab for this assay list
  • The ready-to-use PCR panels, including the redesigned assays, have been updated to v5 PCR panels

The Exiqon v4 miRCURY LNA Universal RT microRNA PCR System reagents will continue to be available in limited quantities during a transition period. To order, please contact our Support Team directly.

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Product Product no. Cat. no. List price:
miRCURY LNA miRNA Custom PCR Assay
Contains forward and reverse primers for 200 reactions
339317 Varies
S Fr. 234.00
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miRCURY LNA miRNA Custom PCR Assays are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.


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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).
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miRCURY LNA miRNA PCR System at a glance.
The miRCURY LNA miRNA PCR System uses one single cDNA synthesis reaction for all amplifications, reducing pipetting and saving time and sample. Two LNA-enhanced miRNA-specific qPCR primers enable highly specific and sensitive amplification and single nucleotide discrimination. The fast and easy workflow takes only 3 hours with minimal hands-on steps.


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LNA-enhanced primers result in greatly increased sensitivity compared to DNA primers.
miRCURY LNA miRNA PCR Primers and commercially available DNA-based primer sets were used for real-time PCR amplification of serially diluted cDNA (106–10 copies). LNA-based primer sets showed superior sensitivity in all cases, especially for AT-rich sequences, such as hsa-miR-155 (61% AT) and hsa-miR-1 (73% AT).
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Detection of differential expression of miRNAs in LCM specimens from tissue FFPE sections.
Areas between 8000 and 13000 μm2 of normal tissue, tumor tissue and tumor stroma were isolated by laser dissection from FFPE sections of human colon (A). Total RNA was extracted and miRNA levels were quantified using miRCURY LNA miRNA PCR (B). Normalized data are shown on a log2 scale as relative expression compared to normal tissue. hsa-miR-103 and hsa-let-7a were used as reference genes.
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Differences in miRNA expression between serum samples.
Normalized expression levels of eight different miRNAs in two serum samples are shown. Total RNA purified from the equivalent of 8 µl serum was used in the RT reaction. hsa-let-7a and hsa-miR-103 were used as reference genes for normalization.
Performance
Unmatched sensitivity
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).

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.
Principle
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.

A unique system for miRNA profiling
miRCURY LNA miRNA PCR Assays offer the best combination of performance and ease-of-use available on the miRNA real-time PCR market by combining universal RT with LNA PCR amplification (see figure Schematic outline of the miRCURY LNA miRNA PCR System). Universal RT makes it possible to use one first-strand cDNA synthesis reaction as the template for multiple miRNA real-time PCR assays. This saves precious samples, reduces technical variation and saves time in the laboratory. Plus, both the forward and reverse PCR amplification primers are miRNA specific and optimized with LNA. This provides 1) exceptional sensitivity and extremely low background, enabling accurate quantitation of very low miRNA levels and 2) highly specific assays that allow discrimination between closely related miRNA sequences.

Coverage
Over 20,000 assays are available covering all organisms in miRBase 20. Over 1,400 assays are fully wet-lab validated for sensitivity, specificity, efficiency and background on both synthetic as well as different biological samples. The remaining assays are in silico-validated using a comprehensive design algorithm that ensures high-quality, species-specific, LNA-enhanced assays with optimal sensitivity and specificity within each organism. This means that several different assays may target the same sequence. Ultimately, the assay for each species is selected based on the genetic background of the organism. If you are working with novel miRNAs, such as from an NGS experiment, custom-designed LNA miRNA primer sets for any miRNA are also available.
Procedure
The miRCURY LNA miRNA PCR Assay system is an miRNA-specific, LNA-based system designed for sensitive and accurate detection of miRNA by quantitative real-time PCR using SYBR Green. The first step of the procedure is universal reverse transcription, followed by real-time PCR amplification with LNA-enhanced primers (see figure miRCURY LNA miRNA PCR System at a glance).
Applications
miRCURY LNA miRNA PCR Assays are used as part of the miRCURY LNA miRNA PCR System for:
  • Mature miRNA quantification
  • snoRNA detection

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