A unique system for miRNA profiling
miRCURY LNA miRNA PCR Assays offer the best combination of performance and ease-of-use available on the microRNA real-time PCR market by combining universal RT with LNA PCR amplification (see figure Schematic outline of the miRCURY LNA miRNA 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.
Over 20,000 assays are available covering all organisms in miRBase 20. Over 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 that ensures 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 miRNAs, such as from an NGS experiment, custom-designed LNA miRNA primer sets for any miRNA are also available.
Reference gene assays for normalization
Nine reference gene primer sets are available, 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 cover a large range of Cq
values, offering the possibility to accurately and reliably normalize across a range of miRNA expression levels.
The control primer sets have been validated as reference genes for the miRCURY LNA miRNA PCR System and work optimally with the miRCURY LNA RT Kit and the miRCURY LNA SYBR Green PCR Kits. It is very important to always confirm the consistent and unvarying expression of a gene in all tissues and under all treatments of an experiment before choosing it as the reference. We recommend that you test a number of reference genes and use software such as GeNorm and NormFinder (part of the GenEx software) to choose and validate reference genes.
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)