This review provides an insight into real-time quantitative PCR (qPCR) assays today.

Using end-user feedback it investigates interest in the targets for qPCR applications; primary application areas for qPCR analyses; the importance of emerging applications; how qPCR assays utilising mastermixes are typically undertaken today; what influences the purchase of qPCR mastermixes and thermal cycling instruments; and the level of automation applied.

It also examines some of the latest vendor offerings in this increasingly competitive and rapidly expanding area, highlighting the latest advances in the qPCR toolbox. This includes the development of new qPCR reagents, fluorescent probes, primers, mastermixes and kits; qPCR labware and consumables; realtime thermal cycling instruments; sample preparation devices and systems; data analysis software; and progress in applying qPCR assays to novel assay application areas.

Real-time quantitative PCR (qPCR) was recently reviewed in DDW (1). This article not only provides an excellent introduction to the evolution of method and the theory behind, it also discusses some of the latest concepts and emerging applications. To complement that review, HTStec recently undertook a qPCR end user survey to understand current practices and preferences in qPCR assays and instruments, and to identify future user requirements (2).

In this article we highlight some of the main findings of the resulting HTStec qPCR Assay Trends market report. The report attempts to put into context some of the latest developments and future expectations for qPCR presented by Kubista1. It also provides an insight into how qPCR assays are typically undertaken today in many labs and what influences the choice in purchasing qPCR mastermixes and thermal cycling instruments. We also examine some of the latest vendor offerings in this increasingly competitive and fast-moving area.

qPCR targets and applications

The main targets for qPCR applications where survey respondents were quantifying nucleic acids today were mRNA (81% measuring) and DNA (76%). These were followed by SNP (43%), miRNA (41%) and cancer (38%) (Figure 1).

The application areas which are the primary focus for respondents qPCR analyses today were gene expression studies (30% using) and molecular diagnostics (25%). These were followed by quantification of single or multiplexed targets (10%), then other areas (7%) and basic research (5%) (Figure 2).

Expression profiling and multi-marker diagnostics were rated as the most interesting new and emerging qPCR application areas. They was closely followed by fast PCR and then more distantly by single cell expression correlation, real-time immune PCR and multiway expression profiling. Least interest was shown for qPCR tomography (Figure 3).

Use of qPCR mastermixes

qPCR reagents vendors typically offer mastermixes today. These contain Taq DNA polymerase in a highly optimised reaction buffer, along with the dNTPs (deoxynucleosides) required to perform real-time PCR reactions. The formulation has usually been chosen to give the user the convenience of a one or two part liquid addition to speed up the PCR setup process, along with providing the confidence that each PCR reaction will be the same.

By far the majority (84%) of respondents preferred pre-optimised qPCR mastermixes, used according to manufacturers instructions. The alternative is purchasing qPCR bulk reagents separately, where the end user would optimise and validate each assay in their own lab.

Mastermixes are, however, expensive and users are keen to utilise every last drop. On average around 8% of qPCR mastermixes are wasted or remain unused from each prealiquoted tube or bottle. Once opened the majority (47%) of survey respondents retain and continue to use a qPCR mastermix for >1 week. On average qPCR mastermixes are held for a mean maximum holding time of 6.2 days (Figure 4).

The majority (42%) store these opened mastermixes at +4°C, in the fridge or on wet ice. Of the remainder 27% store at -20°C, refreeze in separate aliquots, defrost and use aliquots only once. In addition a further 26% store at -20°C, but refreeze the original tube and defrost one or more times when required. Only 5% do not store at all, ie use the entire mastermix immediately upon opening without dilution. Almost certainly some of these storage conditions are contrary to the manufacturer’s recommendations (Figure 5).

Respondents rated expectation of higher quality/ purity as the factor of greatest importance in a decision to purchase a vendor’s qPCR mastermixes. This was closely followed by method (kit) simplicity, then past experience, then application support and then reagent pricing. Of least importance were reduced reaction cycles (Figure 6).

Typical qPCR assays

Survey respondents reported an average of 455 qPCR samples (tubes or wells) processed at a single time (ie per batch) and up to 36,000 qPCR samples processed per lab per year. That is equivalent to 80 qPCR assay batches processed per year. The majority of survey respondents were using the total assay volume range 10-25μL (Figure 7).

Although the average total assay volume for qPCR assays undertaken today (2008) was still quite high at 51μL. Only 15% of respondents were using miniaturised volumes of 10μL or less. The most used assay format by survey respondents for qPCR assays today (2008) was the 96-well PCR plate (59% using). This was followed by the 384-well PCR plate (22% using). All other formats (ie tubes, strips, discs, open and microfluidic array) were only used by a minority (6% or less) of respondents (Figure 8).

Real-time thermal cyclers

Of the real-time thermal cycler technologies available to support qPCR assays, those based on a Peltier Element Thermal Block (that would include most of the plate and tube-based systems) was chosen as the best with respect to the following characteristics: thermal heterogeneity, optical sensitivity, fastest cycle times/protocols, fastest data acquisition, highest throughput potential, and most automation friendly. Only for the characteristic most suited to miniaturisation was a Microfluidic Array (eg Fluidigm) preferred (Figure 9).

Data quality expectations were rated as the factor most influencing their purchase of a real-time cycler. This was closely followed by assay design and data analysis capability/features, then high resolution melt (HRM) curve analysis capability, then running costs (ie qPCR consumables) and then past experience (Figure 10).

Automation of qPCR assays

Only 40% of survey respondents have applied automation to qPCR assays today (2008), this is expected to increase to 70% in the future (2010), and seems a long overdue approach to improving assay quality and throughput.

Of those respondents that have applied automation to qPCR assays today, the majority (35%) have applied it to small scale (<100 samples/batch) automation of nucleic acid extraction and qPCR assay setup, a further 25% have applied it to small scale automation of qPCR assay setup only and 20% to automated sample nucleic acid extraction only. So far only a minority (10% or less) of respondents have attempted larger scale (100s samples/batch) automation of any aspects of qPCR assays (Figure 11).

What’s limiting the use of qPCR today?

The cost of instrumentation was rated as the factor most limiting respondent’s ability to make maximum use of qPCR instrumentation. This was closely followed by cost of consumables, and then consumable quality, and data analysis capabilities. The least limiting factor was available instruments lack 21 CFR part 11 compliance/FDA approval (Figure 12).

Table 1 summarises the main vendor offerings that currently support qPCR assay-related activities.

Latest vendor offerings

The following snapshots provide details of some of the latest progress vendors have made in development of new qPCR reagents, fluorescent probes, primers, mastermixes and kits; qPCR labware and consumables; real-time thermal cycling instruments; sample preparation devices and systems; in applying qPCR assays to novel assay application areas, panels and arrays; and data analysis software.

The need for broader qPCR studies and new applications is driving more advanced software data analysis, streamlining workflow and run times, and focusing research into emerging areas of study. Stratagene QPCR products from Agilent Technologies are focused on these trends to deliver solutions which enable researchers to meet their goals.

The Stratagene Mx3005P with MxPro QPCR software is a flexible five-colour qPCR system suited for gene expression analysis, validation of microarray data, pathogen detection, DNA methylation assays, and chromatin immunoprecipitation (ChIP) studies. MxPro graphical user interface (GUI) provides the capability to combine data from up to 12 separate experiments and perform global data analysis on the entire project. This provides researchers with the ability to expand research studies and generate more statistically powerful quantitative data.

To perform larger qPCR studies, researchers are looking to reduce overall reaction from 2.5 hours down to under one hour. Stratagene Brilliant II Fast reagents maintain superior sensitivity of detection with a rapid cycling protocol to complete runs in 48 minutes. The reduction in reaction time while maintaining performance allows researchers to significantly increase throughput.

In addition to larger studies, qPCR applications are expanding into new areas of research, like the analysis of micro RNA (miRNA) expression. Stratagene High- Specificity QRT-PCR miRNA assays, used for monitoring patterns of miRNA expression in certain cancers, are designed and validated for numerous miRNA sequences. A novel primer design strategy ensures single nucleotide discrimination of miRNA sequences to avoid non-specific detection (Figure 13).

AlphaHelix Molecular Diagnostics (www.alphahelix.com) has invented and developed a new technology – SuperConvection™ – to speed up thermal ramping using an elevated g-force. SuperConvection™ has been implemented in a new real-time PCR instrument – QuanTyper™-48 – to enable rapid and sensitive qPCR. As a result, QuanTyper™-48 typically runs 40 cycles including melt analysis in 15 minutes.

SuperConvection also improves the detection sensitivity since it enables rapid and sensitive PCR in larger reaction volumes. Detection sensitivity for any PCR assay and sample is limited by how much of the sample can be used as template in the PCR reaction.

Typically, a DNAextraction procedure yields 100-200μL of prepared DNA, which is more than can be fitted into a 20-50μL PCR reaction. QuanTyper™-48 allows the use of more of the sample than any other platform by allowing rapid and efficient PCR reactions in volumes up to 200μl. This is particularly advantageous for samples with complex matrices and inhibiting substances in combination with a low number of target molecules (Figure 14).

Capillette® – another invention by AlphaHelix – is a reagent cartridge designed for pre-dispensing complete PCR assays, including primers and probes. Capillette® acts as a PCR tube lid and is emptied by centrifugation and therefore fits particularly well with QuanTyper™-48, where highspeed centrifugation is used to deliver the PCR reagents directly at the start of the run. Capillette® reduces hands-on time for PCR reaction setup from minutes to seconds and, together with QuanTyper™-48, enables qPCR reaction setup and analysis in less than 20 minutes.

Megaplex Primer Pools from Applied Biosystems, part of Life Technologies Corporation (www.lifetechnologies.com) a provider of innovative life science solutions, are designed specifically for microRNA (miRNA) profiling applications such as biomarker discovery. They are designed to streamline the workflow for miRNA analysis, address the needs of researchers working with minute amounts of RNA, and provide broad, upto- date coverage of known miRNAs expressed in biological samples from humans, mice and rats.

Megaplex RT Primers provide a single-reaction solution when preparing cDNA for real-time PCR analysis on a TaqMan® Human or Rodent (mouse and rat) MicroRNA Array. Two Megaplex RT Primer Pools are available consisting of high complexity pools of novel stem-looped reverse transcriptase (RT) primers of up to 381 individual RT primers per pool that reduce the number of RT reactions needed to profile miRNA expression.

The Megaplex RT Primers can also be used with individual human TaqMan® MicroRNA Assays or TaqMan® MicroRNA Assay Sets. An optional step using Megaplex PreAmp Primers can also significantly enhance the ability to detect and profile low expressed miRNAs, using as low as 1ng of input total RNA.

Following reverse transcription using Megaplex RT Primers, Megaplex PreAmp Primers, combined with TaqMan® PreAmp Master Mix, uniformly amplify all miRNAs prior to real-time PCR quantitation using TaqMan MicroRNA Arrays. The TaqMan MicroRNA Array is a 384-well plate technology that does not require robotic setup and yet can easily be analysed by the Applied Biosystems 7900HT Fast Real-Time PCR System (Figure 15).

Beckman Coulter (www.beckman.com) provides qPCR sample preparation systems for different throughput ranges and qPCR instrumentation. Sample preparation includes RNA purification with Beckman Coulter’s Agencourt kits or other purification technologies in addition to qPCR reaction setup. The Biomek range of liquid handling devices can be used to prepare qPCR reactions in 96-well or 384-well formats. Integration of plate sealers with clear seal material provide batch processing and increased walk-away time.

Automation friendly systems such as ABI’s 7900HT or Roche’s LightCycler 480 can interface directly with Biomeks for complete automation. Labware holders and special Biomek steps have been developed to automate sample preparation in non-microplate labware, eg Corbett (Qiagen) Rotorgene. Biomek software provides functionality to optimise all aspects of pipetting such as speed or pipette tip motion. Biomek methods are therefore easily optimised to ensure accurate and precise pipetting and thus yield high quality analytical data.

A ‘Wizard-Style’ software interface simplifies method set up for variable sample numbers coupled with multiple gene targets. Samples IDs can be read from a variety of barcoded primary sample tubes or provided as lists for multiwell sample storage. Sample IDs are traced through the process and are reported following pipetting steps. This data tracking capability combined with an optional controlled access (‘21 CFR 11’) results in the highest confidence in any data generated by the Biomek sample preparation workstations (Figure 16).

Bio-Rad Laboratories (www.bio-rad.com/amplification/) offers a range of new and reliable qPCR instrumentation and reagents that can generate results in as little as 30 minutes. Unlike thermal cyclers from other manufacturers, the Bio-Rad C1000™ and S1000™ thermal cyclers offer multiple block formats including 96-well fast, 384- well, and a dual 48-well fast block that is gradient enabled.

Bio-Rad’s CFX96™ and the recently launched CFX384™ real-time PCR detection systems also offer gradient enabled reaction blocks, which facilitate the rapid optimisation of qPCR assays and the ability to obtain reliable results faster. Precision Melt Analysis™ Software expands the utility of the CFX real-time PCR systems to high resolution melt (HRM) analysis. This technique is being adopted rapidly for high throughput genotyping and epigenetic studies.

Bio-Rad’s new SsoFast™ EvaGreen Supermix, the patented Sso7d-fusion protein technology and EvaGreen dye deliver superior performance in qPCR and HRM. The unique Sso7d-fusion polymerase in this supermix enables fast cycling without affecting PCR sensitivity, efficiency and reproducibility.

The dsDNA binding protein, Sso7d, functions to stabilise the polymerase template complex, increase processivity and provide greater speed and reduced reaction times than conventional amplification protocols. Unlike SYBR® Green I, EvaGreen dye exhibits very low PCR inhibition, which makes it an ideal choice for fast qPCR protocols. It can be used at a high concentration to generate greater fluorescent signals and provide increased sensitivity, making it ideal for several applications (Figure 17).

BioTrove’s innovative OpenArray® platform advances genomic research in a range of lifescience fields, including agriculture, disease research, bio-defence and public health. Its flexible format and nanolitre scale allows for easy adjustment of sample and assay numbers, and enables genomics researchers to generate hundreds of thousands of real-time qPCR data points per day, significantly increasing sample throughput, while decreasing time and cost.

Applications for the OpenArray platform in discovery, validation and molecular diagnostics are being explored in the following areas:

1) Pathogen Detection

For the detection of infectious agents, OpenArray provides an alternative format to capture array technology that permits the amplification of individual nucleic acid targets. This format combines the benefits and throughput of hybridisation arrays with the robustness of PCR assays and provides better analytical sensitivity, better specificity and broader dynamic range over conventional gel-based PCR or hybridisation array technologies.

2) Microarray Validation

Increased throughput of both assays and samples using the OpenArray system allows researchers to validate microarray results on large sample cohorts in a very short period of time. Panels of genes, 10s to 100s or 1,000s, can be run with familiar qPCR chemistries (either probe based or dsDNA dyes).

3) Pathway Analysis

Validated in BioTrove’s laboratories, OpenArray Pathways qPCR assay panels contain a pre-defined selection of genes and gene families involved in commonly-studied diseases and physiological pathways.

These panels, which are available for human or mouse studies, can be used for hypothesis-driven discovery efforts for both drug targets and biomarkers. Researchers can screen up to four samples against 600+ genes grouped around specific research areas, including ADME/Toxicogenomics, apoptosis, cancer, cardiovascular disease, inflammation, GPCRs, kinases, transcription factors and signal transduction (Figure 18).

Enigma Diagnostics develops and commercialises fully-automated qPCR instrument systems. In the fourth quarter of 2008, Enigma launched the FL (field-laboratory) system, which is a single sample in, answer out device. The whole process of sample extraction – purification and concentration – is combined with rapid amplification, fluorogenic detection and automated results calling. Sample extraction is facilitated with a magnetic bead process.

The system has an open architecture that can accommodate almost any bead based on separation chemistry. Extracted nucleic acid is transferred to a proprietary Electrically Conducting Polymer (ECP) thermal reactor. Rapid thermal cycling is complemented by a flexible fluorimeter with multiple excitation sources and six detection channels that acquire multiple wavelengths in parallel. This combination delivers the fastest commercial thermal cycler capable of analysing fluorogenic chemistries in a highly multiplex format.

The lyophilised assays are supplied in a single cartridge containing all reagents and consumables for the process without the need for a cold storage or specialist operator. FL is therefore an ideal solution for non-laboratory based testing. At launch, the market focus is fieldtesting for veterinary pandemics such as avian influenza and defence/homeland security testing.

The Enigma ML (mini laboratory) is a multi-station random-access clinical in vitro diagnostic (IVD) platform being developed for operation by non-laboratory staff in the decentralised and near-patient environments. It performs fully automated sample extraction including directly from a swab, real-time PCR and analysis in less than 40 minutes. The platform has a small footprint, flexible architecture and the connectivity for diverse deployment within the healthcare environment (Figure 19).

The latest trends in qPCR are today focused on reproducibility and proper analysis rather than on high levels of multiplexing or exotic fluorescent dyes. To improve reproducibility Eppendorf (www.eppendorf.com) offers a new series of qPCR consumables. The tube strips and plates of this series have white wells, which show up to 10-fold increased reflection of light and provide a more homogeneous background to reduce artificial differences among replicates.

The corresponding cap strips have an inverted dome to avoid unintended scratching or smearing of the optic window. The inverted dome also reduces the total volume of the well to advance low volume application. In addition to the qPCR consumables, Eppendorf now offers the qPCR instrument Mastercycler ep realplex with a lifetime warranty on the 96 LEDs used for fluorescence excitation. This way, users no longer have to worry about the excitation light source (Figure 20).

Eurogentec (www.eurogentec.com) a fast growing leader in qPCR kits and reagents, has just introduced a new range of highly sensitive FAST MasterMixes for Probe and SYBR Assays delivering significantly shorter run times than standard MasterMixes, without compromising the quality of the results. These kits have been especially optimised for use on FAST Real-Time thermocyclers but are also perfectly suited for regular cycling conditions.

Within this fast-moving qPCR context of significantly increasing throughput and decreasing reaction volumes, it is important to develop faster and more efficient solutions, but the ease of use aspect must also be addressed! In its constant search for innovative solutions, Eurogentec has therefore combined its FAST MasterMixes with its new BLUE inert dye in one single product.

The result is the new FAST BLUE qPCR kit whose clearly visible blue colour enables the status of MasterMix dispensing to be verified at a glance while enhancing performance with white PCR plastics. This ready-to-use, easy-to-see formulated mix, allows users to achieve the fastest and most sensitive results across all different FAST and non- FAST qPCR platforms and avoid aberrant results before they occur (Figure 21).

Finnzymes DyNAmo™ qPCR Kit family features optimised kits for SYBR® Green and probe chemistries for various platforms. DyNAmo™ Flash qPCR Kits allow detection of target DNA with shorter run times in both fast and conventional instruments. All DyNAmo™ qPCR Kits are provided as 2x master mixes. Finnzymes has also developed novel, ultrathin walled (UTW®) reaction vessels for PCR and qPCR. Low-profile UTW® vessels are available in formats compatible with several thermal cyclers and real-time PCR instruments.

In addition to 96- well UTW® microplates, it offers Piko® PCR Plates which represent a completely new plate format. Being a quarter of the size of standard microplates the Piko® PCR Plates provide savings in reagents, materials and space. Piko® PCR Plates are available in 24-well and 96-well versions. When a full conventional 96-well or 384-well plate is not needed, Piko® PCR Plates can be used to save cost and reduce waste (Figure 22).

Fluidigm (www.fluidigm.com) is the inventor and leading provider of integrated fluidic circuit (IFC) technology. The company offers two solutions for quantitative PCR. The BioMark™ system performs real-time and digital PCR. The system incorporates on-board thermal cycling and fluorescence detection. Fluidigm’s new EP1 system provides digital PCR at a very affordable price. For real-time PCR, customers use Fluidigm’s dynamic array. This IFC is available in a 48.48 or a 96.96 version.

Each dynamic array systematically combines samples and assays into 2,304 or 9,216 (respectively) parallel real-time PCR reactions. Microlitre volumes of cDNA from each cell are pooled with probes and primers. Specific genes are amplified and detected. The results are very consistent and reproducible, with a correlation coefficient value of 0.9. You can survey hundreds to thousands of genes with very limited samples. Digital PCR gives a new, exciting and exact method of quantitative PCR. Fluidigm is the only company today offering a commercially proven digital PCR system.

Using a Fluidigm Digital Array, the EP1 or the BioMark systems can easily distinguish between four and five copies and provide results in just a few hours while requiring only minutes of hands-on time. The quantitation in digital PCR is achieved by diluting the starting sample so that there is at most only one molecule in each of 765 wells on the digital array. After amplification, the number of wells containing the product is counted to determine the exact number of molecules in the starting sample (Figure 23).

The Idaho Technology (www.idahotech.com) FilmArray™ integrates sample preparation, PCR amplification, detection and analysis into a highly user-friendly system capable of massively multiplexed real-time PCR. The system contains all necessary reagents freeze-dried into separate compartments in one FilmArray™ test pouch. To begin a run a user simply injects unprocessed sample and water into the FilmArray™ test pouch, and then inserts the pouch into the FilmArray™ instrument.

Through a series of pneumatic pistons and bladders the FilmArray™ instrument drives reagents through micro fluidic channels into reaction blisters embedded in the FilmArray™ test pouch. After extracting and purifying all nucleic acids, the FilmArray™ performs a nested multiplex PCR that is executed in two stages. During the first stage, or PCR 1, the FilmArray™ performs a single, large volume, massively multiplexed reaction. The products from PCR 1 are then diluted and combined with a fresh, primerfree master mix. Aliquots of this second master mix solution are then distributed to each well of an array which is also embedded in the FilmArray™ test pouch.

Each well of this array is pre-spotted with a single set of primers. The second stage PCR, or PCR 2, is performed in singleplex fashion in each well of the array. Reporting of results is based both on real-time PCR curve data and on PCR product melting curve data collected from each well of the array. In summary, the FilmArray™ extracts nucleic acids, tests, and reports results for up to 120 nucleic acid targets in one sample in one hour (Figure 24).

In November 2008 Integrated DNA Technologies (www.idtdna.com) launched its PrimeTime™ qPCR assay products covered by its licence under the Applied Biosystems’ Probe Manufacturing License Program. The PrimeTime™ qPCR mix contains dual-labelled probes and primers normalised and mixed in a more convenient, ready-touse format. Each tube contains enough product to conduct up to 8,000 qPCR assays at a 200nM primer and 100nM probe concentration.

Next generation products will enable end-users to streamline the assay for their protocols, including specification of individual primer/probe concentrations and availability of 96-well or 384-well formats. Near-term plans also include introduction of additional fluorescent dye and quencher combinations to the PrimeTime™ qPCR assay product line. Assays are custom-designed with a validated realtime PCR design algorithm available free online.

This design tool allows the researcher to customise the thermodynamic parameters to match their specific assay requirements, and to incorporate genome and transcript information in the primer and probe design. Researchers can evaluate the resulting primer-probe sets, including; the sequence of both primers and probe, location on the transcript and individual oligonucleotide attributes for each design set – prior to ordering (Figure 25).

Invitrogen has just launched a new versatile dye suitable for gene scanning applications. Gene scanning, a widely used high-resolution melt (HRM) application, searches for the presence of unknown variations in PCR amplicons prior to, or as an alternative to, sequencing. Mutations in PCR products are detectable by HRM analysis by a change in the shape of the DNA melting curves.

When amplified and melted, heteroduplex DNA samples exhibit melting curves with different profiles than those derived from homozygous wild-type or mutant samples. The new EXPRESS SYBR® GreenER™ qPCR SuperMix from Invitrogen has been successfully used in gene scanning experiments (Figure 26).

The SYBR® GreenER™ dye provides greater sensitivity and reduced PCR inhibition compared to other fluorescent dsDNA-binding dyes and as such shows particular utility in HRM analysis. In this example all four assays were able to clearly and correctly differentiate heterozygous samples from homozygous samples in all genomic DNA samples. Overall, this demonstrates the versatility of the EXPRESS SYBR® GreenER™ qPCR SuperMix, which performs reliably in sensitive real-time PCR reactions and HRM gene scanning applications.

Lonza (www.lonza.com) and Bar Harbor BioTechnology have teamed up to bring life science researchers the StellARray™ Gene Expression System. This innovative system consists of GeneSieve™ Query, StellARray™ qPCR Arrays, and Global Pattern Recognition™ Analysis Tool that will enable researchers to perform a complete gene expression experiment.

These three products completely interrelate, allowing a scientist to perform a bioinformatics search to select their gene(s) or pathway of interest, measure gene expression or gene copy number of their samples using Real-Time PCR arrays, and determine the statistical significance of their data using its web-based analysis tool. The StellARray™ Gene Expression System will change the way researchers think about normalisation to housekeeping genes. By allowing the experiment to determine the normaliser, instead of having to choose one a priori, researchers gain novel insight into what their cells are telling them.

These insights are revealed by the Global Pattern Recognition™ (GPR) Analysis Tool, a novel method to analyse real-time (qPCR) data. It permits researchers to identify biologically and statistically significant changes, in some cases less than two-fold, in gene expression that would normally be missed (Figure 27).

The new multiplex-capable Plexor™ technology for Real-Time PCR from Promega uses a novel base pair chemistry. Each target requires only two primers and is measured directly during the amplification process and not through a secondary reaction. The Plexor™ Systems use standard PCR and are based on the interaction between two novel bases. With Plexor technology more than one labelled primer can be introduced into the reaction, allowing multiplexing of real-time PCR.

In multiplex reactions a different fluor is used for each target. Multiplexing can be as simple as combining a target and a control gene in the same reaction, increasing accuracy over separate reactions with the target in one set of wells and the control in another. Multiplexing not only increases accuracy but also saves reagents and plate space, allowing you to assay more samples per plate. Multiplexing can be extended to two or three targets and a control gene. Multiplex qPCR gene expression assays could be used as powerful tools for diagnostic applications.

Plexor™ technology also allows melt curve analysis, which provides an internal control for the final assay design and expedites troubleshooting during development. The Plexor™ Systems are not just a set of reagents – they provide a comprehensive approach to realtime PCR beginning with assay design and ending with data analysis. These systems come with free data analysis and primer design software created specifically for multiplexing. The Plexor™ chemistry is compatible with most real-time PCR instruments available today (Figure 28).

Q Chip Life Science’s proprietary ReaX™ platform, which enables the packaging and stabilisation of biological reagents, has been available as ready to use master mix combinations for PCR reactions. Now ReaX™ offers the first real alternative to lyophilisation as a stability technology uniquely suited to packaging molecular diagnostic and point of care assays. The platform is a unique microfluidic device where multiple reagents can be mixed with an appropriate polymer and formed into beads in precise and defined ratios.

Beads produced by this method are extremely uniform in size and content with less than 2% variation in size. Bead size can be specified from 80 to 2,000μm. Using this platform whole PCR assays can be packaged into a single bead. Each ReaX™ bead contains all the components necessary to perform a PCR assay including probes, primers, enzyme and other components and are usually provided pre-welled into appropriate plasticware (eg 96-well plates, tubes, strips etc). ReaX™ is compatible with most reporting chemistries used for qPCR reactions.

Assays can therefore be stored in a pre-dispensed beaded format ready for immediate use, with beads dispersing within the first few minutes of a thermal cycle. Q Chip has recently developed several proof-of-concept assays for detection of bacterial hospital acquired infections to demonstrate ReaX™’s capability. These include assays for MRSA, Klebsiella and Pseudomonas. Using the ReaX™ format can substantially streamline the workflow of a molecular diagnostic PCR. It does not change the assay dynamics in any way and the beads are fully and irreversibly dispersed within the first few minutes of a thermal cycle programme.

Using the ReaX™ system minimises user input, time and skill level giving accurate and reproducible results. This format easily allows different molecular diagnostic assays to be performed simultaneously. Complex PCR assays can be encapsulated into a single ReaX™ bead including multiplex assays and also those that require internal positive controls. Additionally, reagents for reverse transcription can be encapsulated into a ReaX™ format including both two and one-step ReaX™ beads for qPCR (Figure 29).

QIAGEN (www.qiagen.com) is launching the Rotor-Gene Q cycler and a range of dedicated Rotor-Gene Kits. Ultrafast, reliable results in realtime PCR are assured through the combination of the cycler’s and the kits’ unique technologies. The Rotor-Gene Q is based on a centrifugal rotary design. During cycling, all samples spin rapidly in a chamber of fast moving air past the same short optical path, which is comprised of up to six individual channels.

This results in minimal optical and temperature variation between samples, ensuring highly precise gene quantification and superior performance in optional High Resolution Melting (HRM) analysis. Rotor-Gene Kits contain a readyto- use, optimised master mix specially developed for fast-cycling, real-time PCR on the Rotor-Gene Q. Patent-pending Q-Bond and other master mix components enable extremely short denaturation, annealing and extension times. Due to a proprietary buffer system, no optimisation is required for the wide range of procedures covered by Rotor- Gene Kits.

PCR, two-step RT-PCR, and one-step RT-PCR can be performed, and detection can be achieved using SYBR Green, sequence-specific probes, or multiple sequence-specific probes (ie, multiplex PCR). Rotor-Gene Kits are well suited for gene expression analysis and other applications. Also compatible with the Rotor-Gene Q are various dedicated QIAGEN kits for virus detection, SNP genotyping, DNA methylation analysis, and miRNA detection (Figure 30).

RealTime ready Focus Panels, the first members of the new RealTime® ready product line from Roche Applied Science, are functionally tested, pre-plated, qPCR assays targeting selected genes from specific pathways or functional groups. Each assay contains target-specific primers and a Universal ProbeLibrary® probe, supplied dried down on LightCycler® 480 96- or 384-well plates – just add master mix and sample cDNA.

Focus Panels for Apoptosis, Cell Cycle Regulation, Nuclear Receptors, GPCRs, ABC Transporters and Reference Genes are currently available in LightCycler® 480 multiwell plates. More panels are planned for the future and in addition, customers will have the ability to order customised panels. With the RealTime® ready product line, Roche Applied Science now offers convenient content supply for the LightCycler® 480 platform and in combination with the LightCycler® 1536 Instrument, to be launched in 2009, this opens new possibilities for high throughput quantitative gene expression analysis (Figure 31).

SABiosciences is introducing three new epigenetic PCR Array technologies that take advantage of the quantitative power of real-time PCR to analyse multiple epigenetic markers simultaneously, specifically DNA methylation, chromatin remodelling due to histone modification and microRNA. The Methyl- Profiler™ DNA Methylation PCR Arrays replace the currently used bisulfite-based methods with selective restriction digests to prepare DNA before quantifying methylated and unmethylated DNA by real-time PCR.

Catalogued PCR Arrays profile the methylation status of hypermethylated tumour suppressor gene promoters associated in published studies with specific cancer or tumour types. The ChampionChIP™ PCR Arrays for chromatin immunoprecipitation simultaneously analyse multiple regulatory sites bound by nuclear proteins. One available array format allows researchers to study the interaction of transcription factors or co-regulators with their binding sites on multiple genes. Another array format analyses the distribution of modified histones or chromatin remodelling factors across a 30kb promoter region of any gene.

Building from the RT2 Profiler™ PCR Array concept of combining pathway-focused gene content with SYBR® Green optimised real-time RT-PCR expression analysis, the RT2 miRNA PCR Arrays monitor the expression of up to 376 miRNA sequences having published associations with cancer or development. All of these technologies now easily allow any laboratory with access to real-time PCR to better understand the epigenetic mechanisms regulating gene expression (Figure 32).

Sigma-Aldrich’s newest qPCR reagents, like all of its products, are based on its excellence as a chemical and enzyme manufacturer. However, with the latest generation of products, Sigma’s focus is primarily upon meeting the needs of the end users. First and foremost, today’s qPCR users demand a suite of products that function seamlessly with their instrument of choice.

To that end, it is developing and directly validating reagents on the newest generation of qPCR instruments, including the Applied Biosystems 7900HT and the Roche LightCycler 480 Real- Time PCR Systems. In addition, its reagents are being optimised to support the end users’ need to utilise fast-cycling protocols and their desire to expand the utility of qPCR into areas such as SNP genotyping and plus/minus assays.

Finally, Sigma has begun to address the key need for the training and education required to develop robust, accurate, and reproducible experimental protocols for qPCR. Through a combination of online seminars and onsite wet-lab courses, it is now able to provide end users with the knowledge and the tools necessary to demystify qPCR and improve their own results.

Takara Bio has just announced the introduction of SYBR® Premix Ex Taq™ 2, the newest addition to the Perfect Real Time Series. SYBR® Premix Ex Taq™ 2 includes Takara’s high-performance Ex Taq™ Hot Start DNA Polymerase, SYBR® Green I, and a newly formulated high specificity buffer. It also features antibody-mediated hot start technology to limit nonspecific amplification and primer dimer formation.

SYBR® Premix 2 is compatible with a variety of real time PCR instruments and high speed qPCR applications. Two ROX reference dyes are also supplied as separate components for systems that require an internal reference. In summary, SYBR® Premix 2 provides the extremely high specificity and sensitivity required for accurate quantification over a wide range of template concentrations (Figure 33).

Thermo Fisher Scientific recently launched ABsolute Fast qPCR Master Mixes for rapid, reliable probe-based detection of DNA and cDNA targets. ABsolute fast qPCR Master Mixes have been developed using Thermo-Fast, a novel hot-start Taq DNA polymerase, which requires an activation step at 95°C for five minutes – compared to 15 minutes required for traditional qPCR master mixes.

Further time savings can be achieved by significantly reducing the dwell times at both the denaturation and annealing/extension steps of the reaction, bringing the qPCR protocol down to less than one hour. ABsolute Fast qPCR Master Mixes have been optimised to deliver high performance qPCR results, on all major qPCR platforms without having any adverse effects on the specificity, efficiency, sensitivity and reproducibility of an assay. Furthermore, they contain an inert blue dye that provides visual confirmation of accurate pipetting.

This is ideal for use with white QPCR plates, which reflect increased signal back to the detector for improved assay sensitivity. 2-step QRT-PCR results obtained using ABsolute Fast qPCR Master Mix under fast thermal cycling conditions have been verified as being accurate, highly reproducible and consistent across a particularly large dynamic range (nine orders of magnitude, from 1 x 10-1 to 1 x 10-9 of human cDNA dilutions) (Figure 34).

WaferGen’s SmartChip Real-Time PCR System™ is designed as the first whole genome, high-throughput gene expression real-time PCR platform. This innovative system, combined with next-generation chemistry and optimised assays being developed by WaferGen, promises to deliver significant speed and cost advantages to researchers in the gene expression and genotyping markets. The SmartChip system will be the first platform to combine the highthroughput capability and cost efficiencies of existing microarrays with the sensitivity and accuracy of real-time PCR.

Specifically, the SmartChip platform’s high density, rapid cycling configuration is expected to provide industryleading throughput levels, while offering discovery and validation capabilities in a single step. The result will be the ability to conduct gene expression research at a fraction of the time and cost currently produced by existing instrument systems.

For example, the company anticipates that whole genome assay time may be significantly reduced with the SmartChip system as compared to several days or months with microarrays or real-time PCR, respectively. Additionally, the system will provide a number of key ease-of-use features including content-ready, high-density chips containing 30,000-100,000 nano-wells with gene panels optimised for cancer, toxicology and whole genome. The user-friendly SmartChip Real-Time PCR System will be preloaded with some of the reaction components.

At the same time, the system will only require a very small sample size as compared to other technologies and platforms and will offer real-time detection and sophisticated read-out options while assuring detection sensitivity and temperature uniformity across chips.

Summary

It is evident from the number of vendors that contributed material to this article that the qPCR assay-related space is not only is well supported, but highly competitive and rapidly changing.

The choice of available qPCR reagents, fluorescent probes, primers, mastermixes and kits is ever increasing. A particular focus here has been the development of fast reagents, some based on novel hot-start Taq DNA polymerase, all delivering significantly shorter run times than standard mastermixes, without compromising the quality of the results.

In addition, new dyes are providing greater sensitivity and reduced PCR inhibition compared to older fluorescent dsDNA-binding dyes, and as such show particular utility in HRM analysis. Most products are now optimised and tested to deliver high performance and function seamlessly on all the major qPCR instrument platforms without having any adverse effects on the specificity, efficiency, sensitivity and reproducibility of the qPCR assay.

Innovations in qPCR labware (eg microplates, tube strips and other plastic consumables) include the wider availability of white plastics (eg in microplate wells) which have shown up to a 10- fold increased reflection of light and provide a more homogeneous background to reduce artificial differences among replicates. Inclusion of an inert blue dye within the mastermix to aid visual confirmation of accurate pipetting, also appears to enhance performance when using white PCR plates.

Other approaches include pre-configured microfluidic cards minimising experimental variability and effort required to run 384 qPCR assays in parallel; plastics devices (eg inverted dome cap strips) to reduce the total volume of the well and enhance optical reading; PCR tube lids that can deliver pre-aliquoted reagents upon centrifugation; ultra-thin walled plates to aid rapid heat transfer; and new small plate formats (eg quarter-size plates) providing savings in reagents, materials and space.

Through a combination of new technology approaches, yielding improved instrument performance, and tailored reagents kits, the latest generation of real-time thermal cyclers are achieving ultrafast and reliable results. Five- or sixcolour detection channels that acquire multiple wavelengths in parallel are ideally suited to emerging applications (eg gene expression analysis, validation of microarray data, pathogen detection, DNA methylation assays and chromatin immunoprecipitation studies).

Other features offered include gradient enabled reaction blocks, which facilitate the rapid optimisation of qPCR assays and the ability to obtain reliable results faster. In addition, the first 1536 plate-based qPCR cycler will be available in 2009.

Increasingly, qPCR detection modules are being included with systems designed to fully automate sample preparation or miniaturise assays.

These systems range from:

1) Commercial real-time cyclers interfaced directly with automated liquid handling devices that can be used to set up qPCR reactions in plate or gene-disc formats
2) Standalone turnkey instruments suited for non-laboratory based testing like bio-defence
3) Randomaccess clinical in vitro diagnostic platforms being developed for operation by non-laboratory staff in the decentralised and near-patient environments
4) Fully miniaturised microfluidic array and chip based formats, operating on the nanolitre scale delivering the advantages of speed and cost savings to massively multiplexed real-time PCR

One thing many of these fully-integrated systems have in common is the provision of complete assays lyophilised in a cartridge containing all the reagents and consumables for the process without the need for a cold storage or specialist operator.

Further enhancements in stability technology are enabling multiple reagents to be mixed with an appropriate polymer and formed into beads, which can be pre-dispensed into plates and dispersed with the first few minutes of a thermal cycle. This approach is uniquely suited to packaging in molecular diagnostic and point of care assays.

Judging by the current vendor focus of monitoring patterns of microRNA expression (miRNA) is one the main targets of qPCR assays for use in profiling applications such as biomarker discovery. Others include DNA methylation, chromatin remodelling due to histone modification, to analyse multiple epigenetic markers simultaneously, validation of microarray data, and pathogen detection.

Some of the array-based platforms are being used to screen panels targeting selected genes and gene families involved in commonly-studied diseases from specific pathways or functional groups. Digital PCR is now available in an array format and looks set to impact on next generation sequencing. Emerging qPCR applications are driving more advanced data analysis.

These include software for high resolution melt (HRM) analysis and the use of global pattern recognition analysis tools, enabling users to combine and analyse realtime (qPCR) data from multiple separate experiments to perform complete gene expression studies on their entire project. Many vendors give away free data analysis tools and primer design software created specifically for multiplexing with their reagents. However, all this may not be enough if qPCR is to fulfill its huge potential.

Recently the urgent need for verifiable and stringent quality control checkpoints at each stage of the experimental qPCR workflow was highlighted (3). It appears that at long last some suppliers and organisations (eg TATAA Biocenters) have woken up to the fact that training and education to develop robust, accurate and reproducible experimental protocols for qPCR are badly needed. Providing end users with the knowledge and the tools necessary to demystify qPCR is now a major priority and ultimately it should help improve the reliability and transparency of qPCR derived data. DDW

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This article originally featured in the DDW Winter 2008 Issue

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Dr John Comley is Managing Director of HTStec Limited, an independent market research consultancy whose focus is on assisting clients delivering novel enabling platform technologies (liquid handling, laboratory automation, detection instrumentation and assay reagent technologies) to drug discovery and the life sciences. Since it formation five years ago, HTStec has published 37 market reports on drug discovery technologies and Dr Comley has authored 26 review articles in Drug Discovery World. Further information on accessing the market report ‘qPCR Assay Trends 2008’ can be obtained by visiting www.htstec.com or by emailing john.comley@htstec.com to receive a free copy of the Report’s Executive Summary and Table of Contents.

References

1 Kubista, M. Emerging realtime PCR applications. Drug Discovery World 2008; 9(3): 57-66.

2 qPCR Assay Trends 2008 Report, published by HTStec Limited, Cambridge, UK, October 2008.

3 Bustin, S. Real-time polymerase chain reaction – towards a more reliable, accurate and relevant assay. European Pharmaceutical Review 2008 13(6): 19-27.