March 1, 2007 (Vol. 27, No. 5)
Multiplexing Capabilities and Optimized Primers Enable Next-Generation Expression
Although quantitative PCR has been available for many years, companies are still developing methods to enhance it. Many of these methods will be showcased at the upcoming Cambridge HealthTech “quantitative PCR” conference to be held in San Diego. Several presentations that will be given at the qPCR meeting are reviewed in this article.
One of the challenges in using FFPE (formalin-fixed, paraffin-embedded) tissue samples is extracting RNA of good quality to use for various studies such as gene expression, disease mechanism, or validating differently expressed genes between normal and disease states. “There are millions of blocks of FFPE samples stored around the world,” explains Natalie Novoradovskaya, M.D., Ph.D., staff scientist II, R&D, at Stratagene (www.stratagene.com). “The main question was whether it is possible to isolate RNA.”
Dr. Novoradovskaya says that standard RNA isolation methods do not work. Formalin, used to fix tissue, causes cross-linkage between nucleic acids and proteins, leading to RNA modification and degradation. The company’s Absolutely RNA® FFPE kit offers a protocol for phenol-free isolation of total RNA from FFPE tissues.
The first step in using the kit involves removing the paraffin, then removing proteins by protein digestion, and finally, using high temperatures to incubate the samples. Dr. Novordovskaya says the kit is based on guanidine thiocyananate isolation, which isn’t toxic like xylene and isolates RNA.
Her group tested the kit on FFPE COPD lung tissue samples (5–10 years old). Although the RNA was degraded, it was still available for amplification. After RNA isolation, her group measured the concentration and purity of RNA (contamination may inhibit the reaction).
Samples are then run on the Agilent (www.agilent.com) Bioanalyzer to see the size of the RNA molecules. This is followed by qRT-PCR to measure gene expression. “We were able to get high yields and RNA suitable for qRT-PCR. This study was to confirm that our kit works and that RNA from FFPE samples can be used for gene expression studies,” states Dr. Novoradovskaya.
Increasing Consistency from Sample Prep to Validation
One of the biggest decisions when it comes to qRT-PCR is which technology to use, in terms of either DNA-binding dye or probe technologies. In order to address this, researchers at Thermo Fisher Scientific (www.thermofisher.com) have been developing solutions to improve qRT-PCR reactions. “One of the areas we’ve done a lot of work in is how to prime RNA and what to use, which is dependent on what you are trying to measure in the end,” explains Ian Kavanagh, Ph.D., R&D manager.
“There is not a lot of peer-reviewed data regarding this though, which can have profound effects on your results.” According to Dr. Kavanagh, the main choices for priming RNA include: oligo dT, gene-specific priming, and random priming. However, he says the best way to provide a good starting platform is to use a blend of random priming and anchored oligo dT, which together will prime any type of RNA and provide good sensitivity.
The company has launched a line of new kits, Abgene® Absolute™ Blue qPCR Master Mixes, which feature a colored dye to significantly enhance the contrast between reagent and plastic, without affecting the reaction. “Visualization of the master mix reduces the chance that there will be aliquoting errors, increasing reproducibility and improving consistency among different sample types,” adds Dr. Kavanagh. “We’ve also found a use for it in high-throughput reactions using 96-well plates to check that aliquoting is even across the entire plate.”
Optimizing qPCR Experiments
Scientists at Bio-Rad Laboratories (www.bio-rad.com) have developed methods to enhance the real-time PCR process and will discuss areas that need trouble-shooting. Starting with how to design the assay, Richard Kurtz, Ph.D., product manager, gene expression, explains that two main aspects include primer and amplicon design.
“Primer design is key to having a specific, optimized PCR reaction. Primers must bind target sequences at specific temperatures to prevent nonspecific amplification that negatively affects efficiency.” In addition, he says two main issues when first using real-time PCR include reproducibility and reaction efficiency.
One way to optimize an assay for performance is to use a thermal cycler gradient to ensure optimal annealing temperature for the primers. This enables the user to program the experiment so that each row or column (depending on the instrument) will be at different temperatures during specific steps of the protocol, allowing up to 12 simultaneous experiments. This saves time on finding an optimum annealing temperature to use for the assay.
Another topic covered is optimizing reactions if the user is going to multiplex. “Some of the things you may want to consider include: having a well-optimized single-plex assay and making sure you have the same Ct (cycle threshold) values between your single-plex and multiplex assays,” states Dr. Kurtz.
He says that the majority of customers are using real-time PCR for gene expression in human tissues, plants, and animals. “The big questions from our customers performing gene expression assays revolve around housekeeping or reference genes, which ones to use and how to test them as a reference,” he adds.
Some troubleshooting areas Dr. Kurtz will address include: what happens if there are primer-dimers and how that affects a TaqMan® experiment, what it means if there is poor reproducibility, how to assess a good reaction, and what happens if there is contamination.
Dr. Kurtz says since this technique is relatively new, most users have their own way of setting up experiments. “I’m not sure that will ever get standardized, all the instruments tend to do slightly different things with the data. However, in general, there is a standard way for people to analyze their data, especially for gene expression analysis. What we are finding is that people are going a bit beyond that, so they are looking at things like multiple housekeeping genes and incorporating that into their analysis results and incorporating different reaction efficiencies into results as well.”
One solution for analyzing low-expressing genes, such as G-protein coupled receptors (GPCRs), from limited samples is Applied Biosystem’s (www.appliedbio.com) TaqMan low-density arrays. “This presentation will be focused on GPCRs,” says Raymond Samaha, Ph.D., senior manager, gene expression R&D. “We have a six-panel array of about 380 GPCRs that comes in a microfluidic card (TaqMan® Low Density Array GPCR Panel). We couple this with an amplification approach, TaqMan PreAmp Master Mix Kit, which uses the same primer combinations that exist in the Taqman assay.” The preamp approach allows detection of low-expressed proteins and enables detection of 40% more of these genes.
“It is difficult to obtain RNA from paraffin-embedded tissues since the fixative causes cross-linking and degradation. Typically you get small pieces of RNA that you can’t use for microarrays,” says Dr. Samaha. “But, with the preamp approach, you’re only amplifying a small piece, which is what the TaqMan assay is targeted against, so it gets around the degradation problem.”
Dr. Samaha adds that one of the issues with amplification is not distorting the representation of molecules in the population. If everything is amplified to the same level, the differential expression is lost. “We’ve shown with the preamp approach that if you compare the differences in Cts between amplified and pre-amplified, you always maintain the differential expression between several tissues.”
Bringing Molecular Diagnostics to Market
There are many steps involved in bringing a product through the approval process, including different requirements for ASR, RUO, and IVD products. Walter Koch, Ph.D., vp and head of research at Roche Molecular Diagnostics (www.roche.com), will explain some of the guidelines.
ASRs, or analyte-specific reagents, are components of CLIA-certified lab’s homebrew, laboratory-developed assays. These are developed and manufactured under the Quality Systems Regulation and are listed, rather than approved, by the FDA. They do not contain instructions for use as part of the test and cannot be promoted for specific diagnostic applications.
RUO, or research use only, is a separate designation for kits sold for research only. These contain no performance data in labeling and no “intended use” diagnostic statement.
IVD, in vitro diagnostic, is a medical device, by FDA definition, that analyzes human body fluids to provide information for the diagnosis, prevention, or treatment of disease. The test kit may be a complete system commercialized for a specific intended use and require FDA clearance or approval, depending on the regulatory application made.
Dr. Koch says that, typically, a company like Roche Molecular Diagnostics follows five phases of development. These include: analysis, feasibility, development, implementation, and manufacturing/sales. In his presentation, he will explain some of the difficulties in bringing a project to market.
Further requirements may include establishing clinical utility, educating labs on new technologies, interacting with payers on reimbursement issues, consulting with regulatory agencies for guidance on correct regulatory paths for individual products, and educating clinicians who ultimately order the tests.
Promega (www.promega.com) has developed real-time quantitative PCR systems that are multiplex-capable using base-pair chemistry.
The Plexor™ Systems measure each target directly using the amplification process and not via a secondary reaction. It uses only two primers per target, which makes the design of multiplex assays simpler. “The advantages of multiplexing are huge, including more accurate data, increased productivity, and even increased efficiency of PCR,” explains Ilgar Abbaszade, Ph.D., strategic marketing manager, functional genomics.
The system works by measuring reduction in fluorescence signal during amplification. One of the two primers contains a modified base with a fluorescent reporter at the five prime end.
As amplification proceeds, fluorescence is reduced by site-specific incorporation of a fluorescent quencher, which is attached to the modified nucleotide. “As soon as the quencher is in close proximity to the fluorescent dye, it results in reduction of fluorescent signal,” explains Dr. Abbaszade. “This is the opposite of TaqMan, where the curve increases from low to high.”
After PCR, a melt analysis can be done to provide an internal control for the final assay design or to expedite troubleshooting during development. The system also provides free web-based primer-design software to design primer sets that will work together with maximum efficiency. “The probability of the software not working is low, we get about 92% of the reactions working,” adds Dr. Abbaszade. Plexor technology works on most currently available real-time instruments.