Design LNA™-enhanced qPCR primer sets for any RNA using Exiqon’s Custom LNA™ qPCR Assay design tool. The tool is optimized for RNAs ranging between 50-10,000 bp in length, including mRNA, non-coding RNAs or your own custom sequences. For shorter RNA targets, < 27 bases, we recommend using our custom microRNA design tool.
LNA™-enhanced primers offer several advantages over conventional DNA primers. They increase the flexibility in primer positioning which is particularly important for AT-rich targets. In addition, LNA™-enhanced primers can be made shorter than conventional primers, which is advantageous when working with low abundant targets or targets from degraded/ highly complex samples. The use of LNA™ also allows precise adjustments of primer melting temperatures. Primers designed using our online tool have an optimal Tm for use with Exiqon's SYBR® Green Master Mix.
The easy-to-use design tool accepts sequence, accession or keyword as input. The resulting qPCR assays are displayed graphically for easy viewing and selection. In addition, the tool offers highly developed features, such as automatic handling of introns and variations as well as the ability to design common assays for transcript families. Advanced options let you select specific regions to target, anchor primers or make changes to introns and variations.
As a highly innovative feature, the design tool also links mRNA targets to predicted or validated microRNAs and allows easy ordering of associated microRNA qPCR assays.
Assays designed using the tool are optimized for use with Exiqon’s Universal cDNA Synthesis and SYBR® Green Master Mix kits.
The recommended PCR conditions are the same as for our pre-validated and custom miRCURY™ microRNA qPCR assays allowing mRNA and microRNA to be quantitated at the same time. Please contact us for further recommendations on combined microRNA and mRNA analysis.
PCR primer sets designed with Exiqon’s online software tool are provided in amounts sufficient for 100 or 250 PCR reactions when used with Exiqon’s Universal cDNA Synthesis and SYBR® Green Master Mix kits. Please see the Instructions for LNA™ qPCR for details.
The primer sets can be ordered directly online or by contacting us with the web order reference number (obtained by downloading order as PDF from the shopping basket). Prices can be found by placing the designs in the shopping basket.
In a process which typically takes about two minutes, an advanced algorithm evaluates approximately 2,500 primer pair designs based on more than 45 different criteria to find LNA™-enhanced optimal primer sets for your target sequence.
Note, the design software has been designed for mRNA but can also be used for long non-coding RNA and DNA templates.
The design criteria include:
The design of forward and reverse primers will be specifically adjusted toward each other and is optimized for use with Exiqon's SYBR® Green master mix.
- 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 Exiqon’s vast knowledge of LNA™ oligonucleotide design.
- Sequence database searches to identify potential off-target hits in the transcriptome with high sequence similarity. This ensures that the designed primer sets are highly specific for the target for which they were designed.