October 15, 2009 (Vol. 29, No. 18)

Establishment of Standards and Evolution of Methodologies Help the Field Mature

Quantitative PCR (qPCR) technologies have exploded in the last decade. Commonly used for both diagnostics and basic research, new and exciting advances are being made in such areas as biomarker discovery and treatment monitoring. This rapid growth has also come with some problems, including what to call it. The technology is variously known as real-time PCR, real-time qPCR (RT-qPCR), qRT-PCR, and several other incarnations.

A new movement is under way that provides qPCR guidelines, not only for more consistent terminology, but also for more reliable experimental practice. Additionally, researchers are developing new ways to overcome obstacles in experimental designs and data analysis. Presenters at Select Biosciences’ “Advances in qPCR”, held recently in Berlin, described innovations as well as new strategies for overcoming bottlenecks.

The remarkable success and popularity of qPCR has also come with an assortment of challenges, according to Ivan Delgado, Ph.D., application scientist at Helixis. Dr. Delgado discussed a recent initiative designed to help ensure data relevance, accuracy, correct interpretation, and repeatability.

“In 2008 over 18,000 publications referenced qPCR. While most scientists demonstrate appropriate practice, some have failed to use this technique appropriately. A consortium of well-known researchers developed and recently published guidelines designed to provide a comprehensive toolkit for more uniformly performing and reporting qPCR data. As is often the case, the trick is in the details.”

The so-called MIQE (pronounced mykee) guidelines provide a recommended checklist for performing assays as well as documentation to accompany journal submissions (see August 2009 GEN, page 40).

“MIQE consists of 85 guidelines that can be quite daunting for scientists new to qPCR,” Dr. Delgado reported. “There is no question that many of these guidelines are definitely critical to generating reliable data, yet some are desirable best-practice. The next step for our community is to establish how to integrate these guidelines practically and seamlessly into everyday science.”

Two of the most critical guidelines address assay validation and template quality assessment. “It is important to assess the performance of the qPCR assay itself,” Dr. Delgado said. “For example, what is the efficiency of the reaction, the linear dynamic range, limit of detection, and the precision? qPCR reactions should first be validated by running a standard curve to evaluate the efficiency of the assay. Another important consideration is the need for good quality control of the nucleic acid samples, in particular RNA. Accurate quantification and quality assessments are needed to be sure of the input and integrity of the RNA in the sample.”

Judy Macemon, vp of marketing at Helixis, added that, although the guidelines are voluntary, many in the field believe that implementing the recommendations is good practice. “We are working closely with the guideline developers to make it easier for scientists to adopt them. Our new real-time PCR system software meets all of the MIQE guidelines. And the new Helixis instrument has been designated the first real-time PCR system to provide a MIQE-compliant solution.”

The PIXOTM Real Time PCR system from Helixis

Where Not to Fail

“Junk in, junk out” goes the saying indicating the importance of good starting material. The same applies for data generated by qPCR, according to Ramon Goni, Ph.D., head of the qPCR division at Integromics.

“In our projects, we understood the critical need for checking datasets before they are analyzed,” Dr. Goni said. “Data analysis is only as good as the input data. So, even a correct analysis can produce wrong results if the input data has errors. Our strategy is to provide a workflow that minimizes discretional criteria and human input and instead interfaces directly with instrumentation and provides multilevel analyses.”

One of the company’s flagship products, RealTime StatMiner®, was designed with quality control in mind. It not only integrates filtering criteria, it automatically selects the best endogenous control. “We have designed this solution to allow great confidence in the analysis of experimental data because of built-in quality control measures,” Dr. Goni said.

“For example, the heatmap representation can immediately highlight sample outliers as well as find poor correlations with other samples under the same biological conditions. Also, the user can navigate gene by gene to assess the quality of replicates. It is compatible with all of Applied Biosystems’ RT-qPCR platforms and works with TaqMan® and SYBR® Green studies.”

Dr. Goni presented experimental data at the meeting that described the company’s completed studies utilizing tissue from patients with chronic obstructive pulmonary disease. “It is important to characterize the intactness of patient samples. We utilized RealTimeStatMiner to identify samples with mRNA degradation and outliers in measurements. This allowed us not only to validate the conclusions of the qPCR project, but, more importantly, to help optimize results. We’ve just submitted our data for publication.”

The company continues to expand and refine the software. “After every congress and meeting, we always come up with new ideas and new challenges. Methods continue to change, so it’s important to continually update your product.”

Integromics’ RealTime StatMiner® is a step-by-step guide for qPCR data analysis.

Molecular Beacons

New qPCR technologies involving molecular beacons were described by Fred Kramer, Ph.D., professor at New Jersey Medical School. Like the faithful lighthouses that send out signals to ships at sea, molecular beacons provide biological signals useful in applications that range from rapid pathogen identification to genetic screening.

“We have been developing ways to rapidly identify potentially lethal sepsis-causing bacteria. Identification in a clinical setting often takes several days, during which time a patient is treated with a broad-spectrum antibiotic. This strategy occasionally fails and contributes to the development of antibiotic-resistant strains. With a molecular-beacons approach in a qPCR setting, we can identify pathogens in one reaction in only one or two hours.”

Molecular beacons are single-stranded oligonucleotide hybridization probes with a unique architecture. They form a stem-and-loop structure in which the loop contains a probe sequence complementary to the target. One terminus of the stem contains a fluorophore and the other a quencher. Like a molecular switch, molecular beacons light up only when the probe hybridizes to its target.

According to Dr. Kramer, molecular beacons can be designed to be either “finicky” or “sloppy”. “Finicky probes are shorter (~18–26 nucleotides) and form hybrids with perfect complementarity. These are useful for genetic screening, detection of SNPs, and pharmacogenetic applications. Sloppy probes, however, are longer (~40 nucleotides) and hybridize to a broad range of species we want to identify.”

This feature helps identify pathogens in a clinical sample. “We use a sloppy probe set with four differently colored fluorophores in a single gene amplification reaction,” he said. “The molecular beacons hybridize at a relatively low temperature. As the temperature is slowly raised, each of the four probe-target hybrids melts apart and signal is lost. The resulting set of four melting temperatures creates a specific signature that identifies which of potentially hundreds of species is present in the sample.”

Dr. Kramer has licensed the molecular-beacons technology for an assortment of clinical tests that range from HIV-1 identification to tuberculosis screening. While his current research is still at the development stage, he is optimistic that advanced molecular-beacon designs will enable new clinical diagnostic applications.

Scientists at New Jersey Medical School are using a molecular beacon approach to expedite qPCR research.

FFPE Samples

Another emerging technology that uses modified primers was discussed by Adam Baker, Ph.D., director of diagnostic product development at Exiqon. “It is critical for us to be able to analyze microRNAs accurately and robustly in blood-based and precious clinical FFPE samples. Analyzing microRNAs from these types of specimens can be exceedingly difficult due to low RNA yields and insufficient quality.”

Exiqon’s new qPCR system allows high-quality microRNA profiling to be carried out in samples with low RNA yields. According to Dr. Baker, the new qPCR platform, called miRCURY LNA™ Universal RT microRNA PCR, features two important advancements.

“First, because the system is based on a universal reverse-transcription reaction, the same first-strand cDNA pool can be used as template in all microRNA PCR amplification assays. This means that up to 740 different microRNAs can be profiled from just 40 nanograms of total RNA. Second, the qPCR assays use two microRNA-specific primers containing locked nucleic acids (LNA™). LNA nucleosides are “locked” by a methylene bridge connecting the 2´-O atom and the 4´-O  atom enabling both higher affinity base pairing and enhanced mismatch discrimination.”

Dr. Baker noted that this has allowed the development of exceptionally sensitive and specific assays that enable accurate quantification of low microRNA levels and discrimination between highly related microRNA sequences. “Using this system we have been able to carry out sensitive and robust screening in clinical cancer samples. As a result, we are developing diagnostic tests based on these results. We are gearing up to launch the new assay system this year.”

Detection of differential expression of two microRNAs in laser-dissected specimens from tissue FFPE sections (Exiqon)

Unique Twist with RNase H

Integrated DNA Technologies (IDT) has developed a new system for detecting nucleic acids in complex samples. According to Mark Behlke, CSO, the company is “utilizing a novel system that employs PCR with modified primers that have an internal RNase H2 cleavage site. RNases H are endonucleases that specifically hydrolyze the RNA strand in an RNA/DNA hybrid.

“In this system, a cleavable linkage (RNA base) is positioned near the 3´-end of the DNA primer which is blocked at the 3´-end and cannot function as a primer without cleavage. After the primer hybridizes to its target, the terminal blocking group is removed by the RNase H2 enzyme and amplification occurs. The hybrid is sensitive to correct base pairing so that unblocking is inhibited by the presence of a base mismatch near the location of the cleavage site.”

Dr. Behlke reported that the technology is especially useful for enhancing specificity such as for high-homology gene-expression studies, rapid SNP analysis, and allele discrimination.

Presenting an example that employed SYBR™ Green in the PCR assay, Dr. Behlke described experiments using human or rat cDNA with primers specific for the human HRAS gene, a member of the Ras oncogene family.

“The PCR reactions were run for 60 cycles. Despite the relatively large differences in sequence between the rat and human genes, a false-positive signal was generated in rat cDNA using human-specific primers. When we converted the identical sequences to the blocked primer design and used the same target cDNA samples, the blocked sequences showed the same true positive signal in human cDNA, but did not give any false amplification in rat cDNA. Although we used SYBR Green, it is also possible to use blocked primers labeled with a fluorophore and a quencher. This alternative format permits multiplexing.”

The company expects to release the technology in 2010. It is currently performing beta testing, preparing papers for journal submission, and seeking partners for applications for in vitro diagnostics.

The remarkable progress made in the field of quantitative PCR also has generated some growing pains. As researchers continue to refine and tackle problems, most remain optimistic that the maturing of qPCR technology will involve becoming more rapid, user friendly, capable of higher throughput, and cost effective. 

Blocked/cleavable primers that improve PCR specificity (Integrated DNA Technologies)

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