Bio-Rad this week introduced qPCR assays and panels that have been wet-lab validated to meet the minimum information for publication of quantitative real-time PCR experiments, or MIQE, guidelines.
With the launch, Bio-Rad claims to be the only vendor whose entire portfolio of qPCR assays has been validated to such standards. The company expects the assays, which cover approximately 95 percent of the human transcriptome, to save researchers time and resources by obviating the need to validate assays in their own laboratories.
The new PrimePCR assays are the fruits of more than a year's worth of labor by scientists at Bio-Rad and Belgian qPCR and bioinformatics firm Biogazelle. The two companies last year inked an exclusive agreement, the financial details of which have not been disclosed, to design, optimize, and experimentally validate every primer pair for nearly all the protein-coding genes that make up the human transcriptome.
All of the assays were designed following strict guidelines on maximum transcript coverage, minimal overlap with known SNPs, and spanning large introns, where possible, according to Bio-Rad.
First published in 2009 in the journal Clinical Chemistry, the MIQE guidelines were designed to provide researchers with a roadmap for improving the quality and reliability of their qPCR data.
Since that time, the molecular biology research community has slowly adopted the guidelines, and many qPCR instrument and reagent vendors have done their part to help their customers follow MIQE protocols.
Bio-Rad and Biogazelle have both been at the forefront of that effort. For instance, in late 2011, Bio-Rad published a case study on its website demonstrating how neglecting key steps in the MIQE guidelines could lead to flawed data and erroneous conclusions (PCR Insider, 3/1/2012).
Bio-Rad had by that time also partnered with Biogazelle to offer the Belgian firm's qbasePlus software along with Bio-Rad's own CFX Manager 2.0 real-time PCR experimental setup and data-analysis software to enhance researchers' ability to comply with MIQE guidelines when using one of Bio-Rad's qPCR systems.
Now, with the new assay offerings, Bio-Rad believes it is at the forefront of commercially available MIQE-compliant and fully validated qPCR assays.
"The idea was really, from both [Bio-Rad and Biogazelle], to do it different from what's been done before," Jo Vandesompele, Biogazelle's founder and CEO and one of the original authors of the MIQE guidelines paper, told PCR Insider this week.
"We all know that assays that are either pre-designed or in silico validated, or, to some extent, wet-lab validated, have been around," he said. "There was no need to just copy the effort of what others were doing. Bio-Rad really wanted to do it differently, and I think we have succeeded. After lengthy discussions we decided to do an entire MIQE compliance validation."
Vandesompele, who is also a professor of functional genomics and applied bioinformatics at Ghent University, added that "the level of validation that has gone into these assays is actually beyond industry standards and common research practice."
Each of Bio-Rad's qPCR assays was rigorously evaluated for specificity, efficiency, and sensitivity.
To test assay specificity, for instance, the scientists validated all PCR products from cDNA using massively parallel sequencing.
"That's a level of specificity that has never been achieved before," Vandesompele said. "We actually pooled all the PCR products in different batches from universal reference RNA … and we sequenced them to an average depth of more than 1,000 fold. And we only retained assays that were entirely specific, and did not have any potential mismatch reads in the genome."
Sam Ropp, senior marketing manager of GXD consumables at Bio-Rad, told PCR Insider in an e-mail that "no other vendor has used next-generation sequencing to validate assay specificity before. Therefore they have had no real ability to fully validate the specificity of their assays."
As such, Ropp added, other vendors "sell assays for any gene target that their assay design software generates. In addition, some vendors promote that the use of sequence-specific hydrolysis probes avoids non-specific target detection. Unfortunately, they don't tell researchers that while the fluorescence may be specific, secondary amplification targets can still negatively impact qPCR reactions."
For efficiency testing, company scientists conducted a ten-fold dilution series of synthetic templates for each assay. "Only assays that displayed good linear performance and efficiency were judged to be of sufficient quality," Bio-Rad said in a statement.
Finally, the companies minimized non-specific amplification by redesigning assays with unacceptably high background signal, mostly from primer dimers; and tested the assays using water as a negative control "to show that there were no aspecific signals in a reaction that should be blank," Vandesompele said.
"So looking at cDNA, genomic DNA, a negative control, and the serial dilution series, together with the sequencing, is really a level of wet-lab validation for every single assay that is unprecedented," he added.
Stephen Bustin, professor of molecular science at the Barts and the London School of Medicine and Dentistry at Queen Mary University of London and another original author of the MIQE guidelines, told PCR Insider in an e-mail that he is "certain that these assays will perform well" given the expertise of the involved parties.
"As astonishing as it sounds, it is absolutely true that assays are being sold that have not been validated empirically," Bustin said, adding that the availability of the Bio-Rad/Biogazelle-developed assays "constitutes an important step toward allowing researchers who, for whatever reason, do not want to design their own assays, to comply with the MIQE guidelines."
Bustin, who has written several books on the subject, added that he was "still surprised that people find assay design so difficult;" and that he and collaborators have published step-by-step assay design protocols to meet MIQE guidelines and design assays that "meet their specific needs better than a generalized commercial assay."
Nevertheless, he said that "if a researcher wishes to buy a commercial assay, I would certainly have no hesitation in recommending the new Bio-Rad assays."
Bio-Rad said that the PrimePCR products are available as individual SYBR Green assays, pre-plated pathway- and disease-specific panels, or custom-configured plates. The panels were designed from canonical pathway maps provided by the GeneGo unit of Thomson Reuters, thus "ensuring that the gene assays present on each real-time PCR pathway and collection plate are the most relevant for gene expression profiling based on differential expression studies and the frequency with which gene targets appear in the peer-reviewed literature," Bio-Rad said.
Vandesompele noted that the companies validated assays covering "close to 95 percent" of the human transcriptome.
"Obviously there are a few genes for which Bio-Rad does not have an assay to date, and this is simply because of specificity reasons," Vandesompele said. "For some genes it's simply impossible … to make an assay that is 100 percent specific — so I wonder what other assay vendors do if they provide an assay. Bio-Rad is considering what they will do with these types of genes — whether they will bring out assays and warn the researchers that the assay may co-amplify very homologous genes; or they will not do that because they can't make a perfect assay."
Bio-Rad's Ropp added that the company is confident that even assays that don't meet all the MIQE standards are still the best available assays for those gene targets, due to the fact they have gone through "many rounds of design and wet-lab validation. However, before we will sell them, we want to develop a method of properly informing researchers about any exceptions to our high validation standards so that they can make an educated decision regarding whether the assay will meet their needs."
Ropp also said that Bio-Rad plans to develop fully validated PrimePCR products for other genomes and applications in the future.