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Feature Articles : Nov 15, 2009 (Vol. 29, No. 20)

Improving and Enhancing the Use of Quantitative PCR

New Instrumentation and Assays Drive Research and Diagnostic Advances
  • Kathy Liszewski

Quantitative polymerase chain reaction (qPCR or real-time PCR) is the gold standard for precise monitoring of selected genes. Its keen ability to both detect and simultaneously quantify specific DNA sequences underlies its popular use in diagnostics and basic research. Additionally, coupling qPCR with reverse transcription (RT-qPCR) provides a powerful means to quantify mRNA in cells or tissues.

The field of qPCR is experiencing a surge of interest and rapid expansion buoyed by advances such as instrumentation that pushes capacity to 1,536 wells and optimization-free multiplexing. Select Bioscience’s “Advances in qPCR” meeting,  held recently in Berlin, showcased innovations in the field and provided expert advice for improving and enhancing qPCR.

Most biologic processes comprise a complex maze of interactions. Identifying the key players involved by utilizing real-time PCR can provide a signature of overall activity. “It is important to understand how the combined activity of many genes contributes to pathways related to health and disease,” said Kathy Lee, senior application specialist for genomic assays at Life Technologies.

“Genome-wide transcriptome studies are often employed to generate an initial list of genes that displays differential expression across treated and untreated tissues, or diseased versus normal tissues.”

New attention is being focused on identifying pathway-based biomarkers because they can be important diagnostic tools and predictors of treatment outcomes. “Finding biomarkers involves direct screening of genes from a specific pathway or a set of biologically or functionally related genes that hopefully provides a biological signature. We have developed more than 150 TaqMan® Gene Signature Arrays for human, mouse, and rat in both a 96-well format as well as a 384-well micro-fluidic cards.”

“It is critical that you trust your results,” Lee said. “The TaqMan Arrays allow reproducible and quantitative real-time, high-throughput screening for many samples and genes. Additionally, they allow a much higher sensitivity, specificity and dynamic range than just DNA microarrays.”

Experimental Variability

To achieve robust and meaningful results requires understanding the many sources of experimental variance in the qPCR workflow, reported Raza Ahmed, Ph.D., qPCR product specialist at Agilent Technologies. “With the drive to detect even smaller quantities of sample, it is crucial to address experimental variability at all stages. Many sources of error can appear such as in the experimental design, during sample preparation and purification, in the qPCR reaction itself, and during post-run analysis.

“Know your sample and its complexity,” Dr. Ahmed explained. “The complexity of a tissue affects the quantification results. For example, analyzing mixed cell populations could lead to underestimating the quantity of the sample. Nonpositive cells can dilute target concentration. Also, where a tissue is sampled is important. Thus, you must make sure your sampling does not introduce bias.”

Care should be taken from the very beginning of the process. “The quality of the template and any inhibitors present can lead to failure of detection,” Dr. Ahmed added. “Further, various compounds can be inhibitory such as phenols, cell debris, EDTA, lipids, and high amounts of RNA or genomic DNA. To get around these obstacles, samples need to be rerun at a higher dilution if possible. Including internal controls helps to monitor any sample inhibitors.”

Care should be exercised even during qPCR set up, according to Dr. Ahmed. “Optimize both your forward and reverse primer concentrations. Adjust the buffer conditions if needed. Take the time needed to optimize and validate your assays. The last thing you want is to have low efficiency and high variation. But with careful scrutiny you can produce reliable results.”

Minimizing Variance

Ian Kavanagh, Ph.D., R&D manager of genomics at Thermo Fisher Scientific, also had advice on how to minimize variance. He said a few quick and simple modifications to one’s protocol can minimize many common sources of variance.

“Many things can go wrong with qPCR and there are a lot of choices labs must make to minimize user variance. A user may have to contend with DNA contamination, which can cause false positives since PCR cannot distinguish between cDNA and genomic DNA. An easy fix is to include shrimp nuclease that destroys double-stranded DNA during the reverse-transcription step, but which is inactivated by high temperatures at the conclusion of that step.” 

Another recommendation is to use white qPCR plastics instead of the traditional clear plastics. “Our studies found that use of white plastics can greatly enhance sensitivity, especially at lower limits of detection,” Dr. Kavanagh noted. “Some of the resistance to adopting white plates is that samples may not be as visible in the wells. We recommend use of an inert blue dye added to the reaction and have launched a product line called Thermo Scientific ABsolute Blue that contains this dye in the mix.”

Speed is usually a desirable quality in any scientific experiment. Fast qPCR is a method that reduces the time of the protocol. According to Dr. Kavanagh, “a common misconception is that fast qPCR requires specialized instrumentation. In reality, you can use standard instrumentation with optimized reagents and cycling conditions. The key to success is carefully designing the assay to minimize the secondary structure within the amplicon and to restrict the length to less than 150 base pairs.

“There will always be areas where variability can creep into protocols. While some are hard to get around, others have less complex solutions such as with DNA contamination and fast cycling conditions. Scientists can drastically improve reproducibility of data and increase the confidence in their results by making a few simple changes. Ultimately, that will save you both time and money,” Dr. Kavanagh concluded.

Solving Multiplexing Issues

Multiplex, real-time PCR enables the amplification of two or more targets in a single reaction. “It’s increasingly being accepted as the technology of choice when needing to simultaneously analyze expression of one or more genes of interest with reference genes as well as for pathogen detection,” said Mark Richards, marketing manager at Qiagen.

Richards noted that researchers still face many challenges when trying to optimize multiplex qPCR. “Multiplexing can be tedious and time consuming because of the need to establish optimization procedures such as adjusting enzyme, primer, DNA, and magnesium concentrations in order to provide the best signal-to-noise ratio. Even after this extensive work, many cases still prove disappointing.”

Qiagen has developed a number of technologies for multiplex PCR that eliminate the need for optimization. “The multiplex PCR technology uses a highly stringent hot-start enzyme, an ammonium containing buffer, and a multiplex PCR-enhancing synthetic factor MP that eliminates the need for optimization because it supports macromolecular crowding. That is, it allows efficient annealing of multiple primers under identical cycling conditions. Scientists have shown that they can detect up to seven targets with this technology.”

“With this system, primer concentrations do not have to be optimized. One can use the same concentration of each. Of course, the primers must be suitable and optimized for the sequence, just as in any singleplex assay,” Annette Tietze, Ph.D., senior global product manager for qPCR, added.

RNAi

Another use of qPCR is to assess the effects of gene expression or knockdown. “RT-qPCR is a valuable technique that can be used to ensure genes have been silenced following RNA interference studies,” Sarah Payne, Ph.D., product manager at TTP LabTech, said. This technique can be used to verify that a gene has been silenced following a positive hit obtained from a phenotypic assay run on Acumen® eX3.

“In one study, our instrument was used in a genome-wide siRNAi screen to characterize the implications of knockdown on a particular cell function and identify the genes involved in epidermal growth factor signaling. PCR techniques were used to confirm that the gene of interest had been silenced. This approach resulted in the successful identification of a number of genes involved in this signaling pathway. Ultimately, following up a positive hit identified by Acumen eX3 with RT-qPCR should result in a reliable, more time- and cost-effective method to distinguish false positives from true hits,” Dr. Payne said.

According to Joanne Franklin, Ph.D., marketing manager, the company is currently finalizing a new application for Mosquito®, its automated nanoliter liquid handler. “Mosquito is now being used to add samples and reagents to a PCR plate with a 1,536-well format that is utilized for miniaturized assays. This will result in reducing the costs of consumables and increasing sample throughput significantly.”

Enhancing Throughput

Scant amounts of sample can be a major limitation for deriving consistent and reliable data from qPCR. Increasing cost is another bottleneck. New tools derived from miniaturization and microfluidics are helping to solve these issues.

In olden times (~10 years ago), PCR technology analyzed up to 96 samples and required 20–50 uL reaction volumes. Fast-forward to the current time, a new instrument enhances throughput 16-fold more to 1,536 wells and needs a miniscule 0.5–2 µL per reaction. The LightCycler® 1536 Real-Time PCR system was recently released by Roche Applied Science.

According to Robertus Van Miltenburg, marketing director, “because of its miniaturization and efficiency, the new system is much more efficient in its use of sample and reagents. This is especially important because often only small amounts of precious sample material are available. Another important benefit is that the number of reactions per run can be increased, which both enhances throughput and decreases the need for plate-to-plate comparisons.”

Gudrun Tellman, Ph.D., product manager, described how the LightCycler 1536 System was recently utilized to perform expression profiling of human heart samples associated with cardiac disease. “We provided examples showing verification of microarray data. We obtained the same results when comparing the traditional lower throughput way or via the 1,536 format, illustrating the sensitivity and the reproducibility of our approach. Ultimately, this greatly enhances how much data you can get from your samples.”

The Roche system uses two excitation filters with two detection filters optimized for detecting mono- and dual-color hydrolysis probes as well as green intercalating dyes. The software facilitates easy set up and running reaction protocols related to the automation of high-throughput data analysis work flow.

New and exciting improvements in implementing qPCR are leading the way to advances in diagnostics, basic research, and the search for novel therapeutics. As sensitivity, scale, and scope broaden, most in the field are optimistic about the continued growth and expansion of qPCR technologies. 

Sidebar: Guidelines for qPCR Consistency

The exceptional success and widespread use of qPCR has led to a prodigious number of publications (~18,000 in 2008). Despite its popularity, the technology lacks a consensus on how best to perform and interpret results. A consortium of experts in the field recently provided new guidelines to help deal with this challenge.

The MIQE Guidelines provide the minimum information for publication of quantitative real-time PCR experiments. They cover a host of recommendations such as the need to provide assay details about reagent components, oligonucleotide sequences, assay sensitivity, and efficiency.

According to co-author Tania Nolan, Ph.D., global manager, technical and application support team, Sigma-Aldrich, “Over time scientists have adopted many different protocols and these differences lead to variations in data. It is impossible to interpret scientific findings unless the relevant methods have been clearly explained. The guidelines are directed to anyone performing qPCR and also to those editing peer-reviewed articles where qPCR data is included. In addition, the guidelines have been sent to journal editors and are being adopted by journals.”

Although they are voluntary, the response has been positive, reports Dr. Nolan. “Journals are considering how they can implement them, and companies are responding by addressing how they can enable scientists to include the required information. Most scientists welcome this greater transparency of scientific reports.” 

Sidebar: New Twist on RT-qPCR Sample Prep

Conventional methods for RNA purification typically involve time-consuming steps and require 30 minutes or more to complete. According to Viresh Patel, Ph.D., senior product manager, PCR reagents, Bio-Rad Laboratories, these approaches usually incorporate one or more of the following techniques—organic extraction, magnetic particles, or salting out—and do not always result in purified RNA that is free of contaminating genomic DNA.

“This is particularly problematic for organic extraction of nucleic acids, which requires a DNase step to remove carryover DNA, followed by exposure to heat (~75°C for 5–10 minutes) to inactivate the DNase before proceeding,” explains Dr. Patel.

“Heating RNA has been shown to result in degradation, especially in the presence of Mg++. Column-based methods that include salting out or magnetic particles provide more efficient RNA purification, though researchers still rely on the use of DNases to ensure RNA purity.”

Using Cell Lysates

Late last month Bio-Rad introduced its iScript™ RT-qPCR sample-preparation reagent for isolation of total RNA and for enabling reverse transcription and real-time PCR to be performed directly from cell lysates. Scientists can use the protocol to remove genomic DNA and stabilize RNA in as little as five minutes, claims Dr. Patel.

“We designed [the product] to meet the needs of researchers performing high-throughput gene-expression analysis, gene silencing, and microarray validation,” he says.

Upon exposure of cells to the sample-prep reagent, cell disruption, and lysis occur in approximately 30 seconds with the aid of mild vortexing, notes Dr. Patel in describing the process.

“The reagent also stabilizes RNA and inhibits intracellular ribonucleases. A short, i.e., two minute, high-speed centrifugation yields total RNA that can be collected in the supernatant, while the nuclei containing genomic DNA form a pellet and can be discarded,” he continues, adding that the resulting RNA preparation is ready for use, without further treatment, in subsequent reverse transcription and PCR/qPCR.