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microRNA LNA™ PCR primer sets

Individual microRNA PCR primer sets for extremely sensitive and specific quantification of microRNAs. Both PCR amplification primers (forward and reverse) are microRNA specific and optimized with LNA™.


New Nature Methods paper compares microRNA profiling platforms

In the largest peer-reviewed study of microRNA profiling platforms to date, Pieter Mestdagh and colleagues have compared the performance of commercially available systems in key areas.

Find out which system offers the best performance
  • Unmatched sensitivity- start microRNA quantification from just 1pg total RNA
  • Ideal for samples with low RNA yield such as serum/plasma, FFPE sections and LCM samples
  • High specificity - discrimination of closely related microRNAs and between mature and precursor microRNAs
  • Fast and easy-to-use - simple two-step protocol takes less than 3 hours
  • Full miRBase coverage – microRNA profiling from any organism using the best qPCR system on the market
  • Assays available in tubes or plates with 200 reactions per tube/well or as ready-to-use Pick-&-Mix plates

A unique system for microRNA profiling

The miRCURY LNA™ Universal RT microRNA PCR offers the best available combination of performance and ease-of-use on the microRNA real-time PCR market because it unites two important features (Figure 1):
  • Universal RT – One first-strand cDNA synthesis reaction (or RT reaction) can be used as template for multiple microRNA real-time PCR assays. This saves precious sample, reduces technical variation and saves time in the laboratory.
  • LNA™ PCR amplification – Both PCR amplification primers (forward and reverse) are microRNA specific and optimized with LNA™. The result is 1) exceptional sensitivity as well as extremely low background enabling accurate quantitation of very low microRNA levels and 2) highly specific assays that allow discrimination between closely related microRNA sequences.

Coverage

20,000 assays are available covering all organisms in miRBase 20. 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, ensuring 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 microRNAs, i.e., from an NGS experiment, custom designed LNA™ microRNA primer sets for any microRNA are available .
 

Note:

The microRNA LNA™ PCR primers have been optimized for use with the Universal cDNA synthesis kit and ExiLENT SYBR® Green master mix kit. Use of other reagents will affect the quality of the results.

Click here for information on Reference gene primer sets .


Figure 1 Schematic outline of the miRCURY
LNA™ Universal RT microRNA PCR System
Schematic outline of the miRCURY LNA™ Universal RT microRNA PCR System. (Click to learn more)

Unmatched sensitivity

The exceptional sensitivity of the microRNA PCR primer sets is achieved by combining the following features:
  • Universal RT
  • LNA™ enhanced and Tm normalized primers
This combination enables accurate and reliable quantification of individual microRNAs from as little as 1 pg of total RNA input in the initial first-strand cDNA synthesis (Figure 1).

In comparison to other microRNA real-time PCR systems using either stem-loop (Figure 3) or standard (Figure 4) DNA primers, the LNA™-enhanced primers offer significantly increased sensitivity, especially for AT rich microRNAs.

The low sample requirements also enables microRNA quantitation using total RNA purified from difficult samples such as LCM samples and serum/plasma (Figure 5 and 6).

Fully validated and optimized

All wet lab validated miRCURY LNA™ Universal RT microRNA PCR primer sets have been optimized to be as sensitive as possible. Over 80% of assays detect at least 5 microRNA copies in the PCR reaction (Figure 7). The primer sets have also been validated for specific amplification of the target and for minimal background signal (Figure 8).

The only platform with perfect specificity

The miRCURY LNA™ Universal RT microRNA PCR System is the only microRNA platform with absolute specificity (see the miRQC study published in Nature Methods) and out-performed a competitor probe-based microRNA qPCR platform in specificity tests (Figure 2). Perfect specificity eliminates false positives and ensures only robust, reliable microRNA signals.

Superior discrimination

The incorporation of LNA™ in the PCR amplification primers (forward and reverse) facilitates the design of assays that can distinguish between microRNA sequences that differ by only one nucleotide (Table 1). In addition, the assays can discriminate between mature microRNA sequences and precursor microRNA (Table 2).

Fast, easy and reproducible

Save time and effort in the laboratory with the 3 hour easy-to-follow protocol. By using the same RT reaction as template in all subsequent PCR reactions, the procedure is greatly simplified compared to systems that require microRNA-specific first-strand synthesis. The number of pipetting steps is reduced to a minimum and technical variation is minimized. As a result, it is possible to achieve extremely high reproducibility from day-to-day and even site-to-site.

Note:

The microRNA Ready-to-use PCR panels have been optimized for use with the Universal cDNA synthesis kit and SYBR Green master mix kit. Use of other reagents will affect the quality of the results.

Figure 1 Accurate quantitation from down to 1 pg total RNA starting material
Accurate quantitation from down to 1 pg total RNA starting material. (Click to learn more)

Figure 2 Specificity test alongside competitor probe-based microRNA qPCR system
Specificity test alongside competitor probe-based microRNA qPCR system. (Click to learn more)

Figure 3 Exiqon's miRCURY LNA™ Universal RT microRNA PCR system is at least 10 times more sensitive than a leading competitor probe-based qPCR platform
Exiqon's microRNA qPCR system is at least 10 times more sensitive than that of a leading competitor. (Click to learn more)

Figure 4 LNA™-enhanced primers result in greatly increased sensitivity compared to DNA primers
LNA™-enhanced primers result in greatly increased sensitivity compared to DNA primers. (Click to learn more)

Figure 5 Laser capture from FFPE material
Detection of of microRNAs in LCM specimens. (Click to learn more)

Figure 6 Differences in microRNA expression between serum samples
Differences in microRNA expression between serum samples. (Click to learn more)

Figure 7 Serial dilution of hsa-miR-181a
Serial dilution of hsa-miR-181a. (Click to learn more)

Figure 8 Specific and sensitive amplification of miRNA
Specific and sensitive amplification of microRNA. (Click to learn more)

Table 1 Single nucleotide discrimination
Single nucleotide discrimination. (Click to learn more)

Table 2 Discrimination between mature and precursor microRNAs
Discrimination between mature and precursor microRNAs. (Click to learn more)


NOTICE TO PURCHASER: DISCLAIMER OF LICENSE No license is conveyed with the purchase of this product under any of US Patents Nos. 5,210,015, 5,487,972, 5,804,375, 5,994,056, 6,171,785, 6,214,979, 5,538,848, 5,723,591, 5,876,930, 6,030,787, and 6,258,569, and corresponding patents outside the United States, or any other patents or patent applications, relating to the 5’ Nuclease and dsDNA-Binding Dye Processes. For further information contact the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.
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