• Sensitivity
  • Less than 10 pg total RNA input is needed – or RNA from a single cell
  • Reliable detection of less than 10 microRNA copies
• Dynamic range
  • Accurate quantification over a wide dynamic range of 8 logs
  • Reliable detection of microRNA with high and very low expression from the same sample
• Specificity
  • Single-nucleotide discrimination, enabling specific detection of closely related microRNA family members
  • Discrimination between mature and precursor microRNAs
• Reproducibility
• More than 98 % correlation between assay-to-assay experiments
• Validated microRNA assays
• High-quality LNA™-based microRNA-specific primer sets - all validated
• Fast and reliable
• Simple two-step protocol done in less than 3 hours
• Short PCR primers reduce risk of false positive results from primer-dimer formation
• Possible to scale for high-throughput microRNA analysis
  

The new miRCURY™ LNA microRNA PCR system is a three-component system developed for rapid, accurate and  sensitive quantification of microRNA by microRNA-specific real-time PCR based on SYBR® Green detection. The system comprises:

   miRCURY™ LNA First-strand cDNA kit
   miRCURY™ LNA SYBR® Green master mix
   miRCURY™ LNA microRNA primer sets

View the list of available microRNA primer sets designed for detection of microRNAs annotated in miRBase 10.0 at The Wellcome Trust Sanger Institute.

Fast and robust PCR procedure
The miRCURY™ LNA microRNA PCR system combines the LNA-technology and SYBR® Green detection and the  a simple two-step microRNA-specific reverse transcription real-time PCR protocol for total RNA. As shown in Figure 1, this allows for the use of miRNA-specific primers in both reaction steps to specifically target the mature microRNA of interest: 1) the initial conversion of microRNA to cDNA by reverse transcription taking advantage of the very sensitive and thermostable Transcriptor Reverse Transcriptase and 2) the subsequent real-time PCR amplification reaction. Hence, an initial general unspecific polyadenylation of the microRNA is not necessary.

LNA improves and even rescues the real-time PCR assay
The design of PCR primers for microRNA quantification 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. A risk of this approach is the formation of primer-dimers in the reaction, which would result in high background signal. By using short LNA-enhanced primers risk of primer dimer formation is minimized and highly specific annealing to the microRNA is obtained resulting in extremely sensitive assays with a wide dynamic range. Moreover, the increased binding affinity obtained from LNA-enhanced primers improves the performance of the assays, and in some cases even rescues an otherwise non-performing DNA-based real-time PCR assay (see Fig 2 for examples).

Extremely sensitive microRNA assays with superior dynamic range
Given the design of the short LNA-enhanced primers, the miRCURY™ LNA microRNA primer sets offer extremely high sensitivity enabling reliable detection of as little as 10 microRNA copies in one assay (see Fig 3). An excellent separation of the amplicon of the desired PCR product and the background signal facilitates a dynamic detection range of more than 8 logs. This ensures reliable detection of both microRNA with high and low expression (Fig 3).

Accurate microRNA detection from a single cell
The miRCURY™ LNA microRNA PCR system requires use of minimal total RNA input. As little as 10 pg total RNA or RNA from a single cell is sufficient for reliable microRNA detection using most of the assays (Fig 4).

Reproducible results
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 (Fig 5). Comparison of individual runs performed on independent days for 30 microRNA assays demonstrated an average correlation coefficient of R2 = 0.9865 for all assays.

Compatible with all real-time PCR instruments
The miRCURY™ LNA microRNA PCR system works well on all commonly used real-time PCR instruments. Figure 6 demonstrates similar and sensitive amplification of the same microRNA assays when using three of the most commonly applied real-time PCR instruments: Roche Lightcycler 480, ABI Prism 7500, and MJ Research Opticon 2.