Although PCR technology is nearly a quarter of a century old, scientists not only use it but depend on it, particularly in the areas of gene expression and microarray processes. Researchers discussed the latest advances in PCR at Cambridge Healthtech’s “Quantitative PCR” conference held recently in San Diego.
Microarrays are moving to the fore as the preferred way to perform parallel measurements of relative expression levels of large numbers of genes among different samples. One problem, however, is the fact that microarrays are notorious for suffering from systematic errors that diminish measured expression ratios below their true values. Agilent Technologies (www.agilent.com) has developed a way to use the combination of external spike-in RNA standards and a commercial direct mRNA assay (called the QuantiGene assay) to objectively optimize both microarray production methods and sample processing methods.
“It’s becoming popular and it’s about to become a lot more popular,” said Paul Wolber, integrating manager, microarray QC development at Agilent. “On September 10, a series of papers is going to be published in Nature Biotechnology that does a cross-platform comparison of all the major microarray and confirmation mRNA measurement technologies.””
Agilent is reversing the methods that pharmaceutical companies use to validate a particular target gene. “We’ve adopted a feedback method,” said Wolber. “If quantitative PCR (qPCR) methods are getting a result, we do two things to reduce the numbers of false positives and false negatives from an initial microarray screen.
“First, we validate the microarray results for certain genes via Quantigene under a variety of microarray processing conditions. Since Quantigene has been cross-validated with qPCR, this also serves to validate on that method.
“Next, we look at the behavior of a series of spiked-in mRNAs, which are basically a part of the gene,” said Wolber. “We use a set of targets derived from human adenovirus. We make mRNA synthetically in a traceable manner, then add known amounts of the various targets to real samples. Once again, we observe how close the microarray comes to delivering the known correct result for the spiked-in targets, under a variety of microarray processing protocols. The desired result is to optimize the protocol. The answer you get has to equal what you’ve put in.”
The optimization projects have resulted in substantial improvements in the accuracy of expression ratio measurements and demonstrate the power of orthogonal analytic methods to guide further advances in microarray technology. “There will be plenty of opportunities for mRNAs to help out PCRs,” Wolber added. “What we’re seeing is the co-evolution of two methodologies, where one can be used to help the other.”
Joe Monforte, CSO of testing and discovery at Althea Technologies (www.altheatech.com), noted that, currently, qPCR using real-time methods has one rather severe limiting factor in multiplexing capability. “While it’s conceivable to develop 5-plexes for gene-expression analysis where quantitation needs to be highly accurate, the practical limitation is a 2-plex. One gene plus one control.”
Partnering with Beckman Coulter (www.beckman.com), Althea Technologies developed an alternative strategy that enables high levels of qPCR for gene-expression analysis, called GeXP. “GeXP makes use of a novel, universal priming strategy and end-point PCR to multiplex 20–35 genes in a single PCR reaction,” explained Monforte. “It yields highly quantitative readout using fluorescence-based capillary electrophoresis systems.”
One particular application stands out. “We have several targeted programs in oncology,” Monforte noted. “Gene expression is a beautiful way to differentiate and diagnose in cancer. The problem is that one gene doesn’t give you your answer. Cancer is triggered by changes in multiple genes, and that’s where multiplexing becomes important; you need to be able to measure larger sets of genes and with GeXP, you have the capability of running as many as 35 genes at once.”
And the applications for this system are broad. “We have customers doing gene-expression-based molecular diagnostics, microarray data validation, and molecular toxicology and immune-panel studies. The research applications are all over the map,” noted Monforte.
Quantifying mRNA Expression in Real-time
“Nanogen (www.nanogen.com) is a diagnostic company, and virtually all we do is based around PCR technology,” said Walt Mahoney, vp of R&D at Nanogen.
Nanogen’s MGB Probe Systems can be substituted for any gene expression assay using other probe and primer chemistries. MGB Probe Systems are short, highly reproducible, produce efficient PCR, and generate linear standard curves, which enables sensitive and accurate gene expression quantitation applications.
Our array system, also PCR-based, is a companion technology that permits higher multiplex than convenient by real-time PCR.”
Like many other companies, Nanogen has products in the qPCR arena. “PCR’s been around almost 20 years. Yet, only 15–20 percent of clinical labs employ molecular technologies. PCR has become critical to infectious disease diagnosis. An important area yet to develop will be the application of miRNA as it relates to cancer. Discoveries involving miRNA have developed over the last five years now,” Mahoney said. “miRNAs are very important expression regulatory elements that tie to cancer when these control mechanisms become deranged. Our technology involves looking at miRNAs and discovering how they can be used as diagnostic tools.
“An additional focus is the study of new markers that can provide a risk assessment as to developing disease or disease progression. Prognostics right now is the biggest stumbling block.”
Nucleic Acid Stabilization in qPCR Gene Analysis
To complement its current line of qPCR reagents and Mx™ QPCR Systems, Stratagene (www.stratagene.com) has developed a novel buffer for lysis of cells and stabilization of released nucleic acids. “We’ve created the SideStep™ method to keep RNA stable on the bench top,” said Lee Scott Basehore, senior research associate.
The buffer lyses cells and immediately stabilizes the released nucleic acids for months. Consequently, the lysate contains the entire nucleic acid complement, and both RNA and DNA can be amplified without the need for purification. Additionally, using primers to amplify a single-copy DNA sequence can be used to normalize input cell mass and accurately measure gene expression between samples by qPCR. And because there is no need for the purification step, researchers can move directly to real-time qPCR detection, mRNA extraction and cDNA synthesis.
“There are a lot of applications for this, but siRNA knockdown validation, miRNA detection, and cDNA synthesis—basically anything that is going to be in the gene-expression arena—this is going to be ideal,” Basehore said.
The stabilization aspect is key. The RNA can be left at the bench for up to eight hours without degradation. “This gives the scientist plenty of working time at the bench, and it can be stored at minus 20 and minus 80 degrees Celsius for at least six months before qPCR is performed,” Basehore noted. “RNA in stored cell lystates show little or no degradation by Agilent Bioanalyzer analysis.” Additionally, Stratagene offers a complete portfolio of SideStep products to suit a researcher’s preferred format or application.
True Real-Time PCR
Applied Biosystems (www.appliedbiosystems.com) not only outlined best practices for performing quantitative gene expression, they showed that it was indeed possible to run a PCR batch and get results in the time it took to give a lecture on the subject.
“We demonstrated a run on the 7500 Fast Real-Time PCR system during our lunch-and-learn at the qPCR meeting,” said Laurel Martin, product manager for the 7300, 7500, and 7500 Fast Real-Time PCR Systems. “Real-time PCR normally takes about 90 minutes to do 40 cycles, but we made several modifications to the 7500 Fast system and developed specialized Fast reagents, so now it’s possible to get those same results in 30 minutes without compromising data quality. It is literally possible to do a product demo, including assay setup and results analysis, in the time it takes to give a talk.”
The application Applied Biosystems has in mind is real-time PCR for the quantitation of RNA and DNA. TaqMan and SYBR Green Chemistries are the tools they used for the quantitation of nucleic acids. TaqMan Gene Expression Assays are a comprehensive collection of over 700,000 probe and primer sets for quantitative gene expression analysis of human, mouse, rat, Arabidopsis, Drosophila, C. elegans, and Rhesus Macaque genes. Custom arrays that use verification plating are also available.
“It also makes standardization across labs easier,” Martin said. “If you have assays, instrumentation, and the controls available to you, you have plug-and-play performance for any application, off the shelf and ready for use.”
And gene expression is the arena they have their collective eye on. “qPCR and PCR as diagnostic tools are really gaining ground,” Martin said. “We see its use in cancer and avian flu research really expanding and as more players get involved, we see this as an area in biosciences that is really going to be growing in the next few years.”
Build Your Own Sequence
The scientist has a number of options when choosing methods to probe DNA targets; many use fluorescent-labeled oligonucleotides. Biosearch Technologies (www.biosearchtech.com) has developed a software program called RealTimeDesign to aid with the technical task of selecting and crafting these oligo sequences.
“When a scientist submits a panel of gene sequences to the software’s scrutiny,” said Ben Sowers, research associate, “RealTimeDesign proposes robust assays without additional user expertise, achieving an average amplification efficiency of 99 percent across seven orders of magnitude of the starting copy number.”
RealTimeDesign provides options to all levels of investigator, Sowers noted. “If you’re a novice, access the Express Mode and leave the decisions to the software. RealTimeDesign will automate all steps and present the highest-ranked decisions to the software. Access the Custom Mode and the software gives you full power to adjust design behind the scenes, to view the sequential nature of the program and select alternate high-performing assays.”
Representative user-modifiable parameters include distance between probe and upstream primer, magnitude of misalignments between oligos, concentrations of primers and probe, and stability of annealing across an oligo’s length.
The software’s direct link to NCBI’s databases enable sequence retrieval using accession numbers, as well as BLAST searches for SNP identification. Interfacing with NCBI’s electronic PCR algorithms, it quickly confirms the specificity of one’s RealTimeDesigned assay.
Sowers noted that RealTimeDesign’s comprehensive algorithms represent one of a collection of advancements helping to push the envelope of Real-Time PCR. Improvements to oligonucleotide synthesis chemistry have produced fluorophores, quenchers, and other exotic compounds that can be fed directly into synthesizers for automatic labeling without any benchtop chemistry. Thermal cyclers have followed suit by automating all aspects of sample manipulation: DNA extraction, purification, amplification, detection, and finally data interpretation.
These breakthroughs have illuminated real-time PCR so that its utility is easily recognized. Immediate PCR results are now demanded with a decisive urgency, including pathogen detection in the environment and in food products. Stepping back, this technology resembles a work of science fiction. Load a sample into your mobile PCR device, and you can quickly determine whether that rhododendron is harboring Phytophthora ramorum.
“Biosearch is all about offering an important piece to a larger puzzle and a service to scientists,” said Sowers. “They can come onto our site, plug in their sequence information, and retrieve ready-to-go assay designs—all for free.”