By Meredith W. Salisbury
For people who aren’t working on the cutting edge of the amplification field, it can be hard to get excited about PCR. Hey, it’s understandable for a technology that’s been a mainstay of this community for 20 years. But for people who pay attention to the tool as it continually expands into new research applications, each evolution of real-time PCR is a breakthrough unto itself. Thanks to the versatility of the amplification technology, it makes its mark on virtually each new research field, schmoozing with whole new groups of scientists until they, too, find they can’t live without it.
This has been the modus operandi of PCR throughout its lengthy career. Beginning in 1985 with Kary Mullis’ inspired plan to get strands of DNA to make copies of themselves, the tool has kept up with the times. One major evolution was the invention of reverse-transcription PCR, which allowed researchers to use RNA strands as the template; another was the development of real-time PCR, also known as quantitative PCR because it gave scientists the ability to quantify mRNA. Over the years, various other improvements, large and small, included finding a thermostable enzyme, specialty enzymes for higher fidelity and longer read length, hot start PCR, higher-throughput reactions, and identifying optimal concentrations and read lengths. These technical advances have all served to put PCR in the hands of people for whom it wasn’t previously a viable tool.
These days, PCR — especially the real-time version — continues to extend its reach. According to experts who spoke with Genome Technology, three major areas into which real-time is currently taking hold are in RNAi, applications with smaller sample sizes, and the clinical and diagnostic arena. Beyond that, indicators show that real-time PCR has a whole host of other applications for which it’s just starting to heat up.
Ask anyone who’s running real-time PCR about what they’re doing today that they weren’t doing a year ago, and one term is virtually guaranteed to pop up: microRNAs. Vladimir Benes, who runs a genomics core facility at EMBL, says his lab has seen “microRNA profiling by qPCR, which definitely was not the case even a year ago.”
“Before the RNAi explosion, people were looking at gene expression,” says Tony Favello at Sigma-Aldrich. “Now they’re looking at changes in gene expression and knockdowns.”
But researchers’ yen to quantify their microRNAs wasn’t a technically simple feat for PCR vendors. Weighing in around just 22 bases, microRNAs proved an especially difficult target for amplification and detection. Applied Biosystems, which launched its microRNA assays this spring, finagled the technology to get it to work in this burgeoning field. “We do a little bit of a trick to use the real-time PCR for microRNA quantitation and detection,” says Kathleen Shelton, senior product manager for microRNAs at ABI. The technique relies on an “RT-specific step where we extend the product … [so we are] able to get the specificity, sensitivity, and reproducibility that you get with regular TaqMan.” So far, ABI has microRNA assays out for human, mouse, and rat, with plans to launch Arabidopsis, Drosophila, and C. elegans shortly. Shelton says her team relies on the information deposited in public databases for sources on which to design their microRNA assays. Additional assays will be developed as data from more organisms is deposited in the public domain.
ABI certainly isn’t the only vendor anticipating growth in this arena. PCR experts at Invitrogen, Qiagen, and Sigma-Aldrich all pointed to the same trend in their conversations with Genome Technology.
And microRNAs aren’t the only genetic snippets to benefit from real-time PCR. Dirk Loeffert, director of R&D in the modification amplification technology center at Qiagen, says he sees customers using the technology to evaluate siRNA potency as well. “Researchers are validating more and more of the siRNA knockdown expression by real-time PCR,” he says.
Smaller Samples and Single Cells
As PCR companies have streamlined the sensitivity of their products, customers have been able to use the tool on smaller and smaller samples — down to the point where, today, scientists can use real-time PCR even on the single-cell level.
“People are doing more and more [studies with] pure samples collected by laser-capture microdissection,” says Mikael Kubista at the TATAA Biocenter. “During the last year or so, a number of robust pre-amplification methods have appeared [making] it possible to analyze small sample amounts.”
For the most part, this level of experiment means bypassing the RNA isolation steps, says Ken Rosser at Invitrogen. “Any type of RNA isolation method that you use is going to result in some kind of sample loss,” he says. And for samples as small as his customers want to use, “you really can’t afford to lose any of your nucleic acid because you compromise your sensitivity.”
Criss Walworth, product line director for gene expression assays at ABI, says the company has a product in development right now that’s a “kind of … sample stretcher.” That type of tool, Walworth adds, will be “really enabling for people who are really sample-limited.” Conceptually, of course, requiring less sample for these experiments opens the doors for customers in forensics or clinical research who previously couldn’t make use of real-time PCR because their samples were simply too scarce or precious.
At the extreme, scientists are now focusing all the way down to the single-cell level. That’s been made possible by new kits, many with isothermal enzymes, that work on whole transcriptome amplification, according to Jo Vandesompele at Ghent University Hospital. The idea is to do pre-amplification and a limited number of PCR cycles to prevent the introduction of any bias into the product, he says.
Kubista says this has allowed scientists to find whole new biological phenomena. His team published a paper late last year showing significant variation in the gene expression in individual cells compared to what shows up in expression studies of a cluster of cells. “That’s very important in trying to understand how biological processes are controlled,” he says, pointing out that even a year ago this kind of study would not have been possible.
Diagnostics and the Clinic
Like so many technologies in the systems biology field, real-time PCR is making its way downstream, with recent appearances in animal and human diagnostics. Dirk Loeffert at Qiagen says that veterinary tests in particular have picked up on real-time PCR as a replacement for some ELISA tests. It’s likely that human diagnostics will follow more slowly, as they may be governed by the FDA. In the meantime, he says, the tool will likely be used to study infectious disease and in GMO detection.
“Today it’s mainly used in research and to diagnose infectious diseases,” says Kubista, “but it’s reasonable that in a couple years’ time we’ll see the first diagnostic kits for complex diseases based on expression profiles.”
In fact, a company called Genomic Health, led by Incyte founder Randy Scott, has introduced a breast cancer diagnostic test that analyzes the expression of a 21-gene panel to predict cancer recurrence.
“The application of real-time PCR in the clinic definitely has increasing popularity,” says Vandesompele. What was once a primarily academic tool has been put to use for pathogen detection and, he believes, promises to provide the basis for a host of diagnostics based on expression pattern.
The Exploration Continues
Experts who spoke with Genome Technology highlighted a number of trends in the expansion of real-time PCR. These include:
Epigenetics — Dirk Loeffert at Qiagen says the trend is still forming, but more and more researchers are coming around to the idea of using real-time PCR to study DNA methylation patterns. Reagents still have to be developed to really encourage this field, he says, but technically speaking, real-time is more effective in helping distinguish a specific from a nonspecific methylation event, he says — which could prove a real boon to epigenetics scientists.
Non-DNA assays — “There’s been a slow but steady use of quantitative PCR for assays that aren’t DNA at all,” says Ernie Mueller at Sigma-Aldrich. Scientists interested in detecting antibodies, proteins, and other analytes are starting to tag those with DNA molecules and “piggybacking off of PCR” to identify by proxy their target of interest, Mueller says.
Immuno PCR — This technique, a tool used for protein detection, is on the upswing, says Mikael Kubista at the TATAA Biocenter in Sweden. What was once an esoteric tool has become “an emerging field” in the real-time community, he says.
The rise of multiplexing — Greg Shipley, a research assistant professor at the University of Texas Health Science Center, says multiplexing will become more and more common in the coming years. Many vendors don’t handle anything higher-throughput than a 384-well plate, but “for any sort of normal screening, 1,536 is the standard in the field,” he says, anticipating that as real-time PCR moves further downstream vendors will amp up their plexing to meet this demand.
Nanoscale reactions — Jo Vandesompele from Ghent University Hospital says current PCR protocols use “huge vessels” and need to be pared down to accommodate nanoscale reactions instead. Two companies he’s got his eye on, BioTrove and Fluidigm, have products that seem promising in helping shrink the real-time reaction, he says.
More gene expression — Real-time is no stranger to gene expression, but scientists believe the demand for this application is on track to skyrocket. As more people realize that data from microarrays is not sufficient to detect lower-level changes, predicts Mikael Kubista, scientists will design their expression experiments to include real-time PCR. One such possibility, he says, would be doing a broad scan on a microarray to find a few dozen genes of interest, and then following up on those with real-time PCR. “It’s more sensitive, the reproducibility is higher, and the dynamic range is much larger,” he says.
Model organism studies — Criss Walworth, a product line director at Applied Biosystems, says demand is growing for organism-specific assays, particularly in the comparative genomics field. Coming soon: targeted sets for rhesus and canine, according to Walworth.
Speedy PCR — Researchers are clamoring for the new, fast PCR techniques vendors have released recently. Some vendors have gotten what used to be an hour and a half process down to the neighborhood of 30 minutes. “Now the running of reactions takes less time than preparing your plates,” says Vandesompele.