miRCURY LNA miRNA Custom Probe PCR Assays
For extremely sensitive and accurate quantification of mature miRNA using LNA-enhanced, probe-based qPCR
- Easily design a custom assay for your specific miRNA of interest
- Detect true signals at very low detection range, down to 1 miRNA copy
- Fast and simple workflow that takes 2 hours
- miRNA assay design for any novel miRNA, miRNA isoform or variant
miRCURY LNA miRNA Custom Probe Assays are custom-designed miRNA PCR primer-probe sets that enable extremely sensitive and specific miRNA quantification with the miRCURY LNA miRNA Probe PCR System. Both the forward PCR amplification primer and the quencher-labeled probe are miRNA-specific and optimized with LNA technology. Assays are easily created online using the same robust and optimized algorithm as our predesigned assays. Thus, custom and predesigned assays can be combined and are guaranteed to work.
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miRCURY LNA miRNA Custom Probe PCR Assay
Custom-designed forward primer and probe for any user-defined miRNA target. Premixed assay for 200 reactions.
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Accurate detection even with high RNA background.|Accurate quantitation even with low starting material.|Discriminatory power down to a single nucleotide difference.|Exceptional efficiency across a broad dynamic range.|miRCURY LNA miRNA Probe PCR System at a glance.|
Four miRNA targets were quantified with the miRCURY LNA miRNA Probe PCR System with and without spike-ins of two different amounts of RNA. Negative controls included the spike-in but no cDNA from miRNA‐specific reverse transcription. Cq values from all reactions were highly consistent regardless of the presence of background RNA. |Designed with LNA‐enhanced, miRNA-specific reverse PCR primers and hydrolysis probes, the miRCURY LNA miRNA Probe PCR System delivers highly sensitive results enabling the miRNA detection from just 1 pg starting material. This robust miRNA quantification system shows high performance across a dynamic range that spans most clinically relevant sample types. |The LNA‐enhanced forward primer and probe of the miRCURY LNA miRNA Probe PCR System are short, so both are designed to be target-specific and to not overlap upon binding. This boosts assay specificity to distinguish closely related miRNAs that differ by a single nucleotide. |A single copy of hsa‐miR‐145‐5p is detected with the miRCURY LNA miRNA Probe PCR System and assay performance remains consistent up to at least 1x106 copies of the miRNA, as evidenced by the high correlation between cDNA copy number and obtained Cq values. |The miRCURY LNA miRNA Probe PCR System uses a single cDNA synthesis reaction for all amplifications, reducing pipetting and saving time and sample. A forward PCR primer and hydrolysis probe that are LNA-enhanced and miRNA-specific deliver highly sensitive miRNA quantification and specificity down to a single-nucleotide sequence difference. The fast and easy workflow takes 2 hours with minimal hands-on time. |
Desempenho
Highly sensitive detection of true signals
The exceptional sensitivity of miRCURY LNA miRNA Custom Probe Assays is achieved by combining universal reverse transcription with LNAenhanced, Tm-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 makes 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 reverse transcription as well as optimized probe 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 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.
Fundamento
The design process
The miRCURY LNA miRNA Custom Probe 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-optimized primers and probe for your miRNA within a few minutes. The tool was 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 Assays have been validated using the QuantiNova Probe Master Mix; use of other reagents for qPCR will affect the results.
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 employs 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, intuitive 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.
Procedimento
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.
Aplicações
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
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For highly sensitive, ultrafast real-time RT-PCR detection of miRNAs from exosomes, serum/plasma, and other biofluids
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For highly sensitive, real-time RT-PCR detection of miRNAs using hydrolysis probes
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Mostrar detalhes
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