Scientifically validated assays
Many companies sell “validated primers” for use in real-time PCR. However this phrase has different meanings depending on the company. We have set out our validation process clearly. We hope that you will see the care which we take to ensure the quality and reproducibility of your gene quantification work. PrimerDesign is happy to guarantee the performance of our real-time PCR assays because we take the trouble to rigorously test every single one.
Primer design
We design the best possible primer pairs for each target gene ordered. Good design is the foundation for sensitive and reproducible real-time PCR assays. Many companies use a single primer design algorithm or database to generate designs automatically. At PrimerDesign, basic designs conform to our own proprietary algorithm, but the final selection will be refined by paying individual attention to detail. Click here from more details on our approach to primer design.
Guaranteed performance
Template homology searches following primer design, potential amplicons are aligned (NCBI BLAST server) with the genome for the target species. This is to look for similarities between the target sequence and any closely related family genes or pseudo genes. Where these are found, further alignments are performed to ensure that no cross reactivity with these sequences will occur during target amplification. In this example, only one significant hit is returned from a rat whole genome BLAST.
Priming specificity
The specificity of the primer for the target sequence can only be guaranteed when the primers are tested on a complex biological substrate. Some companies prefer to test their designs on synthetic oligo-nucleotide templates. This however is a poor model for how the primers will perform in a gene quantification experiment. Our primers are tested on the same material that you use in your experiments e.g. total cell cDNA or genomic DNA. This ensures that your experimental results will mirror our test results. SYBR green based detection is used to generate an amplification plot on CACO2 cDNA . The specificity is demonstrated with a post PCR run melt curve where a single peak indicates that a single PCR product has been generated
This figure shows two different assays each detecting a single product:
Priming efficiency
The priming efficiency of the amplification plot is tested using software such as Lin-Reg which looks for an exponential increase in fluorescence at the critical point for quantification (close to the cycle threshold). In this example the Priming efficiency is 1.94 which is close to the theoretical maximum of 2.0.
Probe hydrolysis
Where a probe based format has been ordered, a double labelled reporter probe is synthesised with FAM label as standard and with the very best Black Hole Quencher. We test the probe with the primers for efficient and amplification specific cleavage resulting in a bright, clean and reliable amplification plot.
Packaging
Validated primer sets are packaged and dried in a PCR clean environment before shipping to anywhere in the World and are of course guaranteed to give you great data on the day the assay arrives.