miRCURY LNA miRNA Custom Probe PCR Assays

Highly sensitive detection of true signals

The exceptional sensitivity of miRCURY LNA miRNA Custom Probe PCR Assays is achieved by combining universal reverse transcription with LNA-enhanced, T_m-normalized, miRNA-specific primers and probe. This combination enables accurate and reliable quantification of individual miRNAs from as little as 1 pg total RNA or down to 1 molecule without pre-amplification (see Accurate quantitation even with low starting material and Exceptional efficiency across a broad dynamic range). The sensitivity and quick workflow make the system particularly well-suited for serum or plasma samples and the QuantiNova enzymes ensure highly reproducible results between technical and biological replicates at the first attempt, saving sample material and time.

Compared with other miRNA probe-based PCR systems that rely on only the probe to generate specificity, the LNA-enhanced primer and probe of the miRCURY LNA miRNA Custom Probe Assays are both miRNA-specific and short enough to prevent overlap when binding to the target (see miRCURY LNA miRNA Probe PCR System at a glance), which improves assay sensitivity, specificity and efficiency. Combined with excellent signal-to-noise ratio (see Accurate detection even with high RNA background), this unique feature allows discriminating closely related miRNAs with sequence differences as little as one nucleotide (see Discriminatory power down to a single nucleotide difference).

Validated and optimized

Assay designs (primer and probe) for novel miRNAs are based on a design algorithm created by LNA and assay experts. This comprehensive algorithm evaluates and optimizes 60 parameters that are validated in silico against highly stringent performance criteria to guarantee a design that offers the highest sensitivity and specificity. The outcomes are assays able to detect down to 1 miRNA copy.

Fast and easy

The easy-to-follow, 2-hour protocol saves time and labor. By using a universal transcription as well as optimized RT-PCR protocols, the procedure is greatly simplified compared to other solutions. Furthermore, the cDNA generated in the RT step can be used across the miRCURY LNA miRNA Probe PCR System, allowing you to seamlessly transition from panels to assays depending on your research needs, and saving sample material and time. The number of pipetting steps is reduced to a minimum, which also minimizes technical variation. Assay robustness allows room temperature setup and delayed runs for greater flexibility and use in automation. This simplified workflow coupled to ISO-compliant assay manufacturing makes it possible to achieve extremely high day-to-day and site-to-site reproducibility.

The design process

The miRCURY LNA miRNA Custom Probe PCR Assay design tool lets you easily design highly sensitive and specific LNA-enhanced PCR primer sets and probe for any miRNA not available as a predesigned assay. The advanced algorithm evaluates approximately 3,000 primer pair and probe designs based on more than 60 different criteria to find LNA optimal primers and probe 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 and probe 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 and probe based on our vast knowledge of LNA oligonucleotide design.
• miRBase searches to identify potential miRNAs with high sequence similarity within the same species. This ensures that the primer sets and probe are highly specific for the small RNA for which they were designed.

A unique system for miRNA quantification

The miRCURY LNA miRNA Probe PCR System enables sensitive, specific miRNA quantification and profiling via real-time PCR. Detection is based on a quencher-labeled hydrolysis probe. The system covers all steps involved in converting miRNA to cDNA and subsequent qPCR detection of miRNAs. The miRCURY LNA miRNA Custom Probe PCR Assays have been validated using the QuantiNova Probe Master Mix; use of other reagents for qPCR will affect the results.

RNA Spike-In Kit for RT

The RNA Spike-In Kit enables quality control of the RNA isolation, cDNA synthesis and PCR amplification steps of miRCURY LNA miRNA Probe PCR experiments so you can identify any poor-quality samples early in the process and save time and reagents. The kit includes UniSp2, UniSp4 and UniSp5 RNA Spike-in Template Mix and the cel-miR-39-3p RNA Spike-in Template.

miRCURY LNA RT Kit

One universal reverse transcription kit for two detection systems. The miRCURY LNA RT Kit is used for both the miRCURY LNA miRNA PCR System that uses SYBR Green for detection, and the probe-based miRCURY LNA miRNA Probe PCR System. The kit includes two system-specific reaction buffers and all the necessary reagents to perform fast and convenient miRNA polyadenylation and reverse transcription in a single reaction step.

miRCURY Probe PCR Kit

Optimized and dedicated for the miRCURY LNA miRNA Probe PCR System, this kit contains the high-performance QuantiNova Probe Master Mix and the miRCURY Probe Universal Reverse Primer. Combined with a miRCURY LNA miRNA Probe Assay or a miRCURY LNA miRNA Probe PCR Panel, the chemistry enables miRNA detection via quantitative real-time PCR.

miRCURY LNA miRNA Probe Assays

These primer and probe assays use miRNA-specific hydrolysis probes and miRNA-specific forward primers designed to detect mature miRNAs using the most up-to-date sequence information from miRBase 22. The miRCURY Probe Universal Reverse Primer is contained in the miRCURY Probe PCR Kit.

miRCURY LNA miRNA Probe PCR Panels

Ready-to-use, predesigned, single-use PCR panel plates to rapidly profile mature miRNAs focusing on the miRNome, specific pathways, certain sample types like serum/plasma, or in a user-designed custom panel.

GeneGlobe Data Analysis Center

This complimentary, web-based analysis tool guides you step-by-step via an easy wizard through the normalization and analysis of your data and generates publication-ready figures.

The first step, the reverse transcription reaction, is an easy-to-perform, single-tube reaction. Total RNA, including miRNA, is used as starting material and the resulting cDNA is used in the real-time PCR amplification with LNA-enhanced primers and hydrolysis probe to detect the mature miRNAs. This enables straightforward miRNA quantification and profiling, accessible to any lab with a real-time PCR cycler.

The miRCURY LNA miRNA Custom Probe Assays are used as part of the miRCURY LNA miRNA Probe PCR System for:

• miRNA expression detection, profiling and quantification
• miRNA biomarker development – screening and identification
• miRNA regulation and pathway analysis
• miRNA detection in complex sample types and samples with low miRNA expression levels (e.g., exosomes, serum, plasma, urine, FFPE sections)
• snoRNA detection
• Validation of miRNA sequencing