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 on the microRNA PCR market by combining universal RT with LNA PCR amplification (see figure Schematic outline of the miRCURY LNA miRNA digital 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.
The principle of the dPCR reaction in the QIAcuity Nanoplates is described here.
Reference gene assays for normalization
Five validated reference gene primer sets are available for dPCR, enabling high-quality data normalization and generation of reliable data from many different sample types (see table below). These control primer sets target endogenous, small non-coding RNAs that are constitutively and relatively stably expressed across different human tissues (see figure Expression levels of reference genes in different human tissues). The control reference genes offer the possibility to accurately and reliably normalize across a range of miRNA expression levels. For more information refer to the miRCURY PCR Controls page.
List of available reference gene assays
|Control primer set, SNORD38B (hsa)*
|Control primer set, SNORD44 (hsa)
|Control primer set, SNORD48 (hsa)
|Control primer set, SNORD49A (hsa)*
||U49; U49A; RNU49
|Control primer set, SNORA66 (hsa)
|Control primer set, 5S rRNA (hsa)
|Control primer set, U6 snRNA (hsa, mmu)*
||hsa, mmu, rno
|Control primer set, RNU5G snRNA (mmu, hsa)*
||mmu, hsa, rno
||Rnu5a; U5a; Rnu5g
|Control primer set, RNU1A1 (mmu, hsa)*
||mmu, hsa, rno
|Control primer set, SNORD65 (mmu)
|Control primer set, SNORD68 (mmu)
|Control primer set, SNORD110 (mmu)
* Validated for use in digital PCR.