microRNA (miRNA) Primer Sets from Exiqon

Sensitivity - Start microRNA quantitation from just 10 pg total RNA
Dynamic range - Accurate quantitation over a range of 8 logs
Specificity - Specific discrimination of closely related microRNAs and between mature and precursor microRNAs
Reproducibility - Highly reproducible data with over 98% correlation between experiments
Fast and reliable - Easy two-step protocol takes less than 3 hours
Validated microRNA assays - Fully validated, high-quality LNA™-based microRNA-specific primer sets

Features

The miRCURY LNA™ microRNA PCR System enables extremely rapid, accurate and sensitive quantitation of mature microRNAs annotated in miRBase. The system combines the unique advantages of LNA™ technology and the well-known SYBR ® Green detection technology in a simple two-step procedure designed for total RNA samples (Figure 1).

First, the microRNA in the total RNA sample is converted to cDNA by reverse transcription using a sensitive, thermostable reverse transcriptase and a microRNA-specific primer. Then, the cDNA is amplified by real-time PCR. The use of LNA™ nucleosides makes it possible to build microRNA-specific primers in both reaction steps, thereby enabling highly specific targeting of the mature microRNA of interest.

Coverage

A variety of high-quality microRNA primer sets are available. To find a particular primer set, please use the search function below. You can search by entering a microRNA sequence, product name or number. For advanced searches, see the search tips below.

All microRNA primer sets are validated for optimal performance ensuring low background and high specificity and sensitivity. Newly validated microRNA primer sets are added continuously.

Note

The Universal PCR primer for the real-time PCR step of the protocol is supplied with the miRCURY LNA™ SYBR ® Green Master Mix.

In addition to this product, a miRCURY LNA™ microRNA real-time PCR assay also requires the following reagents:

Click to view information on Endogenous control primer sets

Figure 1

miRCURY LNA™ microRNA (mirna) PCR System procedure overview

miRCURY LNA™ microRNA PCR System procedure overview. (Click to learn more)

Sensitivity

The miRCURY LNA™ microRNA PCR System is optimized for microRNA quantitation from as little as 10 pg total RNA, which corresponds to the RNA from a single cell (Figure 1). The system can reliably detect 10 microRNA copies (Figure 2).

The design of PCR primers for microRNA quantitation presents a challenge due to the short size of the microRNA target. Standard PCR protocols applied to microRNA would require primers to be full length compared to the target microRNA. The risk of this approach is the formation of primer-dimers in the reaction, which would result in high background. In addition, it may not be possible to make assays sensitive enough for AT rich microRNA sequences. The incorporation of LNA™ nucleosides makes it possible to make very short and yet microRNA-specific primers without sequence overlap, thereby minimizing the risk of primer-dimer formation. LNA™ containing primers also enable amplification of sequences with low GC content (Figure 3). This results in extremely sensitive assays with a wide dynamic range of up to eight orders of magnitudes (Figure 2).

Specificity

Although the LNA™-based primers are relatively short, they still maintain high sequence specificity. A mismatch between the microRNA target and the short primer will greatly affect the Tm of the resulting duplex, thereby also affecting the amplification of the template. The difference in amplification between a perfectly matched and a mismatched target is, therefore, increased by the incorporation of LNA™ nucleosides in the PCR primers. The increase in Tm results in higher binding affinity and higher specificity. By using microRNA-specific primers in both the reverse transcriptase and the amplification steps, Exiqon has created a system with superior specificity and the ability to distinguish between microRNAs that differ in sequence by a single nucleotide (Table 1) and between mature and precursor microRNAs (Figure 4).

Reproducibility

A simple protocol and validated high-quality microRNA primer sets allow for generation of highly reproducible results when testing assay-to-assay variability and robustness (Figure 5). A comparison of individual runs performed on different days for 30 microRNA assays resulted in an average correlation coefficient of R2 = 0.9865 for all assays.

Instrument compatibility

The miRCURY LNA™ microRNA PCR System works well on all commonly used real-time PCR instruments. Figure 6 demonstrates the cross-platform reproducibility observed when three of the most commonly used real-time PCR instruments are compared: the Roche Lightcycler 480, the ABI Prism 7500, and the MJ Research Opticon 2.

High-throughput quantitation and validation studies

The incorporation of LNA™ monomers in the primers enables Tm -normalization of all microRNA primer sets. This makes it possible to quantitate multiple microRNAs optimally in the same experiment for high-throughput screening. The miRCURY LNA™ microRNA PCR System complements miRCURY LNA™ arrays making the PCR system ideal for validation of microRNA profiling data. A comparison of relative fold changes in microRNA expression between two breast cancer cell lines and two prostate cancer cell lines shows an excellent correlation between results obtained using the miRCURY LNA™ Array V11.0 and the mirCURY LNA™ PCR System (Figure 7).

Positive control assay

A primer mix targeting 5S rRNA is provided with the miRCURY LNA™ SYBR® Green Master Mix . Used in combination with the random hexamer provided in the miRCURY LNA™ First-strand cDNA kit , the 5S rRNA primer mix constitutes a positive control assay that can be used for multiple purposes such as assisting in assay setup and troubleshooting.

Endogenous control assays

A panel of endogenous control primer sets targeting selected small nucleolar RNAs (snoRNAs), U6 snRNA and 5S rRNA are available. These primer sets can be used to normalize the microRNA quantitation data from a miRCURY LNA™ microRNA PCR assay.
View more information on our miRCURY LNA™ Endogenous Control Primer Sets .

Figure 1

Accurate microRNA quantitation from a single cell. (Click to learn more)

Figure 2

Accurate quantitation over a range of 8 logs. (Click to learn more)

Figure 3 LNA improves microRNA PCR performance

LNA™ improves microRNA PCR performance. (Click to learn more)

Table 1

miRCURY LNA™ microRNA PCR offers superior mismatch discrimination. (Click to learn more)

Figure 4 LNA improves microRNA PCR performance

The miRCURY LNA™ microRNA PCR System offers discrimination between mature and precursor microRNAs.
(Click to learn more)

Figure 5 Reproducible and robust system

LNA™ gives reproducible and robust results. (Click to learn more)

Figure 6

Compatible with all real-time PCR instruments. (Click to learn more)

Figure 7

An excellent correlation between microarray and real-time PCR data (Click to learn more)

"the scientists at Exiqon were very, very supportive"

Dr. Dorina Veliceasa, from the department of Urology at the Northwestern University Feinberg School of Medicine, discusses the use of the miRCURY LNA™ microRNA PCR system in her investigation of tumor angiogenesis in endothelial cells. Dr. Veliceasa discusses the challenges of qPCR monitoring of microRNA species, including the importance of endogenous controls, and offers advice to other researchers interested in this method of determining microRNA expression levels.

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"The miRCURY™ LNA microRNA PCR system is easy to use and provides high sensitivity and reproducibility.”

"It makes my validation faster, easier and more quantitative"

Scientist Louisa Cheung, Karolinska Institute, Sweden