qPCR Evolving to Meet Emerging Needs

Method Now Utilized in Epigenetics Research and Characterization of Circulating Tumor Cells

Quantitative polymerase chain reaction (qPCR) is one of the most powerful and sensitive means to measure gene expression. The ability to generate data in real time revolutionized the way PCR quantifies DNA or RNA.

Inconsistency of methodologies continues to plague the field, although the MIQE guidelines have helped by defining the minimum information needed to accurately evaluate qPCR. Nonetheless, this bustling field continues to improve and make steady progress. At BioEPS’ recent “QPCR” conference in Germany, advances in assay design and data interpretation were highlighted, and new applications ranging from cancer diagnostics to genotyping featured.

Real-time qPCR is often combined with reverse transcription to quantify cellular messenger or noncoding RNA. But it can also quantify a targeted DNA, an emerging application for the new field of epigenetics.

?“Assays for DNA methylation and histone modifications typically are used to assess epigenetic processes that control gene expression,” explained Viresh Patel, Ph.D., marketing manager, gene expression division, Bio-Rad. “This regulation is mediated through chromatin state changes. Actively transcribed genes have open or accessible, chromatin regions, while genes that are silent are in closed, or inaccessible, chromatin.”

Bio-Rad recently introduced its new EpiQ™ chromatin analysis kit, a tool for researchers who want to assess epigenetic regulation of genes using cultured cells. Dr. Patel said the kit is fast and easy.

“There are two challenges facing epigenetic researchers: time to results and the large amount of input sample needed for analysis. We developed this kit as a new type of assay that interrogates the functional state of chromatin inside the cell. It is easy to perform, quantitative, and produces results on the day of cell harvest. Basically, one lyses cells (as few as 50,000 cells), digests the chromatin in situ, purifies and then quantifies the DNA, and performs the qPCR reaction with specific primers.”

According to Francisco Bizouarn, international field application specialist, the company validated its assay by analyzing 15 genes in 4 human cancer cell lines in which they assessed 4 housekeeping genes and 11 genes that are epigenetically regulated.

“Our data discovered that contrary to the popular notion that gene promoters exist entirely in only open or closed chromatin states, there are intermediate states of chromatin accessibility, too. So there is a continuum from 0 to 100%.”

The EpiQ chromatin assays will be especially useful to complement existing datasets such as for typical assays for DNA methylation and histone modification. “The assays are also very useful for a scientist who wants to determine if epigenetics might be involved in the regulation of a specific target. All one needs is a primer set for target genes of interest and the kit.”

According to Bio-Rad, its EpiQ kit provides quantitative information about chromatin accessibility, which correlates very strongly with gene expression.

Hidden Inhibitors

If you are not already testing for inhibitors in your PCR reaction, this may be the reason you see variable results,” warned Melissa Kelley, Ph.D., research scientist, Thermo Fisher Scientific. “One problem is that routine methods such as spectrophotometric determination of RNA quality can’t rigorously detect the presence of inhibitors such as EDTA, phenol, heparin, and ethanol. A second problem is the common misconception that inhibitors affect different gene targets to the same degree. Both of these issues can produce hidden sources of errors and cause inaccurate or incorrect data.”

The company has designed its Solaris RNA Spike Control Kit using an exogenous RNA control to identify common inhibitors. “A common technique used by researchers is the serial dilution method. But this requires more sample template and more wells—about 15–18 additional wells per assay. There are other kits currently available that use RNA or DNA spike-in molecules. But these may be less sensitive to inhibition because they are added at later steps in the workflow. We performed many optimization studies to determine where to add in a spike control and found that it was best to add it directly to the RNA sample and then do the cDNA synthesis step.”

Reducing the number of samples added to the reverse transcriptase qPCR (RT-qPCR) better conserves limited RNA samples. “For example, you may only have a small amount of patient blood, so the RNA is limited. Our kit requires only six to eight additional wells for each RNA preparation,” Dr. Kelley said.

While the kit doesn’t eliminate inhibitors, it provides valuable information up front. “Determining the presence of inhibitors helps the researcher identify early on if this is a problem. Then you can institute corrective measures to rescue the experiment (e.g., by diluting the RNA) or to re-extract using a better method. Once you have a reproducible method that has the least amount of carry-over inhibitors, you don’t necessarily have to assess for reaction inhibition every time.”

The Thermo Scientific Solaris RNA Spike Control Kit assesses the effects of inhibition throughout the entire RT-qPCR workflow. Points at which a spike-in or exogenous control molecule can be added to a two-step RT-qPCR process are displayed. Inhibitors are most concentrated in extracted RNA and are gradually diluted out during each step (represented by dark blue liquid becoming lighter). A control added to a qPCR reaction will not facilitate detection of inhibition occurring prior to this step.

Predesigned Assays

Designing a good qPCR assay requires careful consideration of a number of parameters that can spell the difference between success and failure, according to Mark Behlke, M.D., Ph.D., CSO, Integrated DNA Technologies (IDT). “The design of the assay must carefully determine parameters such as primer placement, specificity, avoidance of single nucleotide polymorphisms, oligonucleotide interactions, and accurate melting-temperature calculations.”

One answer is the use of predesigned assays that shift the burden of these choices to the manufacturer. But not all assays are created equal, according to Dr. Behlke. “Unfortunately, databases are dynamic, not static. Some commercial assays were designed using now-outdated databases. In other cases, a scientist might be using a primer design format that was done with older, less accurate melting-temperature algorithms. Even worse, a researcher may not be able to find out from the company the sequence information that could be verified or even a correct quote for a research publication.”

To overcome this problem, IDT offers its PrimeTime® predesigned qPCR assays. Scott D. Rose, Ph.D., director, molecular genetics, reports, “We’ve developed these assays for all genes in the human, mouse, and rat transcriptomes. The assays are very specific and eliminate concerns such as cross-reactivity within each genome and primer interactions. Importantly, we also freely provide all the information a researcher will need to know, including full sequence disclosure of both primers and probes as recommended by the MIQE guidelines.”

The IDT technology employs its ZEN double-quenched probes. Dr. Rose indicated that “these assays provide sensitivity down to 10 copies or less. We were able to reduce background fluorescence by decreasing the length between the reporter dye and the internal quencher to only nine base pairs. Traditionally probes have 20–30 bases between the dye and quencher.

“Thus, the ZEN quencher is always within close proximity of the reporter, resulting in a much lower initial background fluorescence, Lower background results in greater sensitivity for the assay. With the ZEN system, even long probes—up to 50 base pairs—are effectively quenched because the distance between the quencher and the reporter dye is fixed and is independent of probe length.”

According to Integrated DNA Technologies, PrimeTime assays are more sensitive than competitors’ assays. A comparison of a competitor’s WDR3 assay and the equivalent IDT PrimeTime predesigned qPCR Assay is shown for five 10-fold dilutions of the target.

Circulating Nucleic Acids

Analysis of nucleic acid fragments present in plasma, serum, and other body fluids such as urine can enable specific detection of tumor types from a simple blood sample, said Martin Horlitz, Ph.D., senior scientist, Qiagen. “These circulating DNA and RNA fragments can originate from malignant tumors, a developing fetus, and also from viral or bacterial infections. Assessing the presence of these nucleic acids via qPCR could help diagnose cancer at an early stage or assess the genetic makeup of a developing fetus.”

Circulating cell-free nucleic acid fragments have distinctive properties. “They are present in blood mostly as shorter fragments of less than 500 base pairs or as nucleotides at a concentration of about 5–100 ng/mL. Because of their low concentration and small fragment sizes, they present a challenge to isolate. Qiagen offers the QIAamp Circulating Nucleic Acid Kit for the quick and efficient purification of these circulating nucleic acids from samples up to 5 milliliters.”

The kit is based upon the selective binding of nucleic acids to a silica-based membrane that provides for improved recovery of fragmented nucleic acids. Dr. Horlitz explained, “The purification is highly efficient with reproducible yields and can be easily done on a vacuum manifold. There is no organic extraction or ethanol precipitation needed. Thus, there is complete removal of contaminants and inhibitors.”

However, even with nucleic acids efficiently isolated, running a qPCR presents another challenge. “Concentration and size distribution of isolated nucleic acids can vary between samples and even in the same individual when samples are collected at different times. Hence, the use of internal controls is recommended to independently track the success of both nucleic acid extraction and PCR quantification.”

Qiagen offers the QuantiFast Kits for singleplex and multiplex real-time PCR and RT-PCR, both of which are SYBR Green and probe-based. “The QuantiFast Kits enable fast and reliable quantification of fragmented circulating nucleic acids of differing sizes and low concentration levels.”

According to Dr. Horlitz, “qPCR of circulating nucleic acids will likely have an impact on molecular testing. While it is already used for certain molecular diagnostic applications, it is still in its infancy. But it will have many applications that range from genotyping to routinely checking for the presence of tumors.”

Targeting Circulating Tumor Cells

Circulating tumor cells (CTCs), released into the blood from primary and metastatic tumors, are rare but important. Scientists believe that CTCs may provide answers not only in understanding their role in establishing metastatic tumors, but also in revolutionizing cancer patient care. Caifu Chen, Ph.D., senior director of genomic assays R&D, Life Technologies, said, “Scientific communities and the biotech industry are racing to develop a novel device to accurately count and molecularly characterize the CTCs so that doctors can treat cancer patients early, individually, and, therefore, more effectively.”

According to Dr. Chen, CTCs have unique signatures in mutation and gene-expression profiles. “We’re developing a new TaqMan® assay technology called RT-qCastPCR, which is capable of digital counting and cancer mutation detection of rare CTCs directly from blood.”

This technology is based upon castPCR, a homogenous assay that combines competitive allele-specific TaqMan qPCR with an allele-specific minor groove binder as a blocker to suppress nonspecific amplification. “We have already validated castPCR assays for a hundred cancer mutations. Some assays can detect one to five mutant molecules in a million copies of wild-type background. CastPCR assays for 44 mutations of KRAS, EGFR, and BRAF genes were released commercially for early access in April.”

Even though the new RT-qCastPCR technology is still at an early stage, the company believes that digital counting and mutation characterization of CTCs in blood could significantly improve CTC research. “This provides direct analysis of CTCs in blood, more sensitive and specific CTC counting based on multiple markers, mutation profiles of every detectable CTC, and a simple TaqMan workflow with answers in a few hours rather than days, as is currently on the market.”

mRNA Expression Profiling

Sabine Lohmann and colleagues from Roche Pharma and Ludwig-Maximillian University discussed the use of customized, function-tested RealTime ready assays for gene-expression analysis in biomarker research and early drug development. They noted that the investigation of mRNA expression profiles in these two areas is of particular interest.

According to the scientists, RealTime ready custom panels offer a variety of intron-spanning RT-qPCR assays covering the major signal transduction pathways, including those relevant for oncology research. The custom panels are composed of ready-to-use LightCycler® 480 multiwell plates containing preplated qPCR assays for human, mouse, and rat targets that have been selected by the user.

The research team explained that they have established various workflows applicable for gene-expression analysis in biomarker research as well as in preclinical and early clinical studies. To illustrate, they said that preclinical drug development was started by profiling gene expression using a multiparameter panel (93 target and 3 housekeeping genes) covering the pathway of interest in a broad screening approach. The goal was to generate a first hypothesis for predictive and pharmacodynamic markers, as well as to provide a workflow with the highest convenience and throughput for this screening approach, they added.

This first step was performed in vitro with tumor cell lines treated with the compound of interest. A workflow protocol was developed, starting with an automated RNA extraction on the MagNA Pure LC instrument in combination with pre-plated RealTime ready customized assays in a 384-well format. The relative gene expression already had been analyzed. After selecting a set of parameters, this first hypothesis was verified using an in vivo mouse model. Xenograft-derived fresh frozen tissue samples were used to test for selected biomarkers.

The team next moved to early clinical development using the first set of clinical samples, formalin-fixed paraffin-embedded (FFPE) tissue derived from various tumor entities. They explained that tumor-derived FFPE tissue is the most relevant sample material for the development of therapeutic compounds in clinical trials or in biomarker research.

RNA extraction from FFPE tissue (e.g., 10 µm sections) using the High Pure RNA Paraffin Kit was followed by qRT-PCR analysis. Then the researchers combined this workflow with function-tested RTR assays.