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qPCR Grows Up

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It's been 26 years and one Nobel prize since the light bulb went off over Kary Mullis' head to usher in the age of PCR. An offshoot of that research-changing approach, quantitative PCR, is reaching its stride in its own right. qPCR has grown up and come into its own as a gold standard in quantifying gene expression.

In the decade or so since qPCR was introduced, it has been streamlined and has become a mainstay in many molecular biology labs. "It is very fast, cost-efficient, and easy to handle, while being a very reliable and sensitive method," says Soroush Sharbati from the Freie Universitaet Berlin. "The development of fast and space-saving cyclers and dropping cost prices contribute to the fact that qPCR is widespread in the molecular biology field."

Quantitative, or real-time, PCR keeps getting better with tweaks to its chemistry to make it faster, programs to help pick better primers, and simplifications to the procedure. Though it is commonly considered the gold standard for gene expression quantification, qPCR still suffers from a lack of proper normalization techniques and standardization. Now, however, researchers and companies are calling for certain minimum standards to be followed in qPCR experiments and are also looking for ways for qPCR data — as the pile keeps growing taller with all the multiplexing — to be more easily shared amongst researchers. Despite those drawbacks, qPCR is just getting started, and its next target is the clinic. There, researchers hope that it can be applied to diagnose disease and even stratify patients for more directed therapy.

"We used various PCR techniques prior to the real-time and they were a big advance on what had gone before, but the real-time version is much more reliable, accurate, and reproducible. It has so many advantages," says Jeremy Garson at the University College London Medical School. "The dynamic range is extremely broad for real-time, and that's particularly useful for virus detection and quantification because the range of concentrations that you get in patients ranges over many orders of magnitude."

qPCR is a-changin'

Compared to the early days, qPCR is much easier to use now. Instead of creating their own master mixes, choosing primers half-blindly, and opening up their tubes halfway through the reaction, researchers can now buy reagents off the shelf and use a software program to help decide what primers are the proper ones to use, while the process has become automated. "I guess it's fair to say it's just that much easier now," says Jon Sherlock, a product manager of TaqMan Arrays and Gene Signature Plates for Applied Biosystems Genomic Assays at Life Technologies. "We have more than 1.2 million pre-designed assays. People can just pick and choose from off-the-shelf reagents." With prêt-à-porter reagents, uniformity and robustness are added benefits.

Roche's vice president of global research, Walter Koch, agrees, adding that scientists have more options these days. "That makes it a lot easier to set up new assays and run them together without having to spend as much time optimizing each and every assay like we used to have to do in the past," he says.

The chemistry of those reagents has also improved and gotten faster — reactions take half the time that they used to. According to Sherlock, the changes to the enzymes, probes, and quenchers all work together to make the qPCR reaction a faster and more robust one. Richard Kurtz, Bio-Rad Laboratories' amplification marketing manager, adds that the fast chemistry reagents have changed the game dramatically. Bio-Rad recently launched SsoFast, one of its next-generation reagents. Kurtz says it's significantly faster as it decreases the time of the annealing and extension stages. What normally would take 15 to 30 seconds is now done in two to five.

Fast chemistry has been changing the reaction across the board. "Another big evolution has been the use of fast chemistry — being able to do the reactions in a fraction of the time and decreasing time to results, increasing throughput," Sherlock says.

The throughput hasn't begun to hit its limit, adds Koch. Well plates have evolved from 24, 96, 384, and now are approaching 1,536. "There's nanoscale opportunities for companies like Fluidigm and others that can potentially go another order of magnitude higher," he says, adding that companies can automate so that a single sample can have a number of reactions run from it. For the researchers, access to bigger plates means using less sample and reagents while increasing sensitivity.

"There is a clear trend for going to ever decreasing reaction sizes and ever decreasing run times as well," says Ghent University Hospital's Jo Vandesompele.

At the same time, qPCR reactions have benefited from better knowledge and annotation of the biology. Primer design software can aid the search for specific primers and many of the programs, such as Vandensompele's RTPrimerDB, are readily available online. "It's the upfront knowledge that has increased specificity, not the actual qPCR reaction itself," says Sherlock. He adds that though there have been improvements to instrumentation and optics as well as tweaks to the chemistry, "in essence, the qPCR reaction hasn't actually changed in all this time." Instead, the researchers' expertise has changed. For example, they now know more about the sequence they are designing primers against, which helps them avoid erroneous hybridization.

It's certainly a check in the 'pro' column that qPCR has remained straightforward to use. The disadvantage that some researchers have become increasingly vocal about is that qPCR can seem deceptively simple.

Living up to standards

Being straightforward to use is a blessing and a curse. Because it's PCR, everybody knows the basics — you just need your target, your primers, and some master mix to throw in the machine. However, the intricacies of qPCR can be overlooked.

Stephen Bustin from Barts and the London School of Medicine and Dentistry says that unless standards are followed faithfully, data can be used to show anything. To highlight that, he points to the now-disproven studies from Andrew Wakefield and his colleagues that showed an erroneous link between the MMR vaccine and autism. "I was so angry about the way the RT-qPCR data had been applied to try to link the MMR vaccine with measles and autism that I felt we really need to make a stand here and make people aware of the fact that this can't go on the way it's been going on," he says. Bustin is now at the forefront of a movement to get researchers to follow a set of guidelines, the minimum information for publication of quantitative real-time PCR experiments, or MIQE, that were published online at Clinical Chemistry in February.

"In my talks, I always refer to the cowboy stage of qPCR. For quite a while everything went," Bustin says. In particular, he casts a critical eye on how people have been normalizing their gene expression data. In northern blot and standard PCR experiments that didn't give quantitative data, people often used a single reference gene. "People just moved that approach to qPCR without thinking about what they were doing," Bustin says. "Are these reference genes really invariant or are they changing with treatment?"

The MIQE guidelines ask researchers to think their experiments through and to be as transparent as possible. The checklist says that essential information, such as the name of the kit used for DNA extraction, the complete reaction condition, and qPCR analysis program, should be included when the study is published. Other information should be included if known, such as volume of the samples, evidence for optimization, and power analysis. Disclosing the probe sequence is "strongly encouraged" though the authors note this is not always possible as some vendors do not provide that information to users.

Another effort for standards and transparency in qPCR experiments is taking on the data format. Many researchers cannot look at their colleagues' data because they use different instruments and analysis software. Ghent's Vandesompele says that a universal data exchange format is sorely needed. "Almost 40,000 papers in the biomedical literature are using real-time quantitative PCR and it's ever increasing. It's almost as exponential as the PCR reaction itself," Vandesompele says. "The problem is that we cannot analyze or reanalyze our collaborators' or our colleagues' or peers' work." To go along with the MIQE guidelines, they've come up with a universal data format called RDML that will allow researchers to share their results and "speak the same language." That language and reporting guidelines are in the April issue of Nucleic Acid Research.

Target: clinic

Despite the issues surrounding normalization and standardization of qPCR experiments, the technology is marching headlong into the clinic. "For pathogen diagnosis, it is valid, if done properly," Bustin says.

The demand from the clinic is on the rise. "We are seeing more real-time PCR being used in the clinical diagnostic setting," Bio-Rad's Kurtz says.

Already, qPCR is hard at work diagnosing viruses. In April, the US Food and Drug Administration issued an emergency approval for a molecular diagnostic assay to identify cases of H1N1 swine flu — and the approach used was real-time PCR. The test used is an rRT-PCR Swine Flu Panel with a CDC assay and runs on Applied Biosystems' real-time PCR machine. "The Applied Biosystems 7500 Fast and Fast Dx real-time instruments have been authorized by the FDA for emergency use in diagnosing swine influenza A using the CDC's specified test at the CDC-qualified laboratories," Sherlock says. "Clearly there's so much confidence in qPCR now that scientists can move beyond the research labs into regulated environments."

The same approach was also used in England. At the end of April, Jeremy Garson said that three positive cases of swine flu had been identified in England using a real-time approach from a routine flu assay. Garson often uses qPCR, replacing tissue culture and immunofluorescence, to identify respiratory viruses including influenza A and B but also RSV, meta-pneumovirus, parainfluenza type I, II, and III, and adenovirus. A few years ago he used qPCR to identify SARS. "It's quick and relatively easy to develop and introduce new assays once the expertise is present in the laboratory," Garson says.

His focus isn't limited to respiratory disease; Garson also looks at other viruses, particularly in bone marrow transplant patients, hepatitis patients, and HIV patients. "One of the key roles is in monitoring antiviral efficacy, " Garson says. "We can determine if [the patients] are non-responders or sustained responders or transient responders and we can modify therapy according to the response."

For some new assays, there are not always standards against which to compare or to determine the significance of a patient's viral level — unlike HIV and hepatitis B and C, for which there are international and national quantification standards. "In some situations, we don't yet know the clinical implications of a certain level," Garson says. "Because the assays are so sensitive, there is the danger of worrying the patient unnecessarily unless quantitative results are available. In most instances, real-time qPCR allows us to discriminate confidently between clinically insignificant low levels of virus and potentially serious higher levels."

Academicians aren't the only ones eyeing the clinic. Roche's Koch says they are interested in moving qPCR into oncology. "I don't think we've begun to exploit … all the ways you can use it to guide differential diagnosis as well as therapy selection," he says. Last year, he says, the big news was that the KRAS mutation could help guide EGFR therapy; he notes that there's a "host of genes that are commonly mutated in common cancers that impact how a patient is going to respond to therapy." Now, he adds, it's practical to stratify patients and optimize therapy.

The future of qPCR most likely holds more of the same as its recent history. "More, faster, quicker, better," says Life Technologies' Sherlock.

Bustin adds that due to all its advantages, the number of qPCR papers is growing exponentially. "I'm very confident about the future of qPCR for the next five years, certainly, and probably longer," he says. "I think that's why it's important that we start getting our act together."

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