qPCR: Current technologies and future applications

A diverse and widely applicable laboratory technique, qPCR is vital for the progression of drug discovery, enabling detection and quantification and commonly used for both diagnostic and basic research. This roundtable brings together experts from a wide range of pharmaceutical applications to discuss current technologies and future applications of qPCR for drug discovery and the pharmaceutical industry.

1. What technologies that are currently available are most effective in delivering results when conducting research in this field?

Nour: “Omics (genomics, transcriptomics, proteomics, metabolomics and phenomics) disciplines were recently specialised and extensively used for drug discovery. Proteins remain an imperative target directly involved in drug development because disease processes and treatments often manifest at the protein level. It is known that more than 80 per cent of pharmaceutical drugs act through proteins. In this field, techniques are mainly based on protein separation. Techniques developed in the late 1970s, like the 2-Dimension (2-D) gels, or newer techniques like Mass Spectrometry (MS), field-flow fractionation (FFF), microarray protein and capillary electrophoresis are the tech – nologies of choice when applied to drug discovery. The question that we can ask is “what about Quantitative PCR (qPCR) and its application in this field?” It is surprising that the most used technology in genomics or transcriptomics has no equivalent in proteomics, hence qPCR is used in gene expression and DNA detection and quantification.”

Huggett: “There are a number of available technologies that are equally effective at delivering good quantitative PCR (qPCR) data and the choice of which very much depends on the required use, which can be highly diverse; for example, a high throughput virology laboratory has very different requirements from a machine than a small research laboratory that is investigating RNA splicing. Furthermore, this can affect considerations about upstream technologies, like robotics, that may be used to facilitate pre-PCR processes. This is reflected by the fact that you will find people preferring certain instruments, but this is usually a personal preference. The real trick to delivering effective results when conducting research withthese techniques is to ensure that experiments are carefully designed and conducted, as outlined by the Minimum Information for Publication of Quantitative Real-Time PCR Experiments; the MIQE guidelines (Bustin et al, Clin Chem. 2009 Apr;55(4):611-22.).”

Pfaffl: “My research focus is in molecular physiology and molecular endocrinology of farm animals. My key technology for expression profiling and to reliably quantify microRNA and mRNA is the real-time RT-PCR, and it will be my choice for the future. Small studies will be carried out in single one-step qRT-PCR assays. Expression profiling in medium throughput (96- to 384-well) will be done on validated qPCR arrays. Either the arrays were assembled with own established SYBR assays or they will be purchased and severely validated by us before usage.”

2. What factors are currently driving the qPCR industry forward?

Huggett: “Microfluidics, high throughput and the associated automation are possibly some of the key areas of hardware development driving the industry forward. Newer technologies that allow automated processing and simplify the data analysis will go hand-in-hand with the above. From the diagnostic side, while equally affected by the above, the concept of point of care (POC) is also undoubtedly influencing the qPCR industry and while a dipstick type POC test is possibly a bit much for this tech nology, the idea of near patient instruments that do not need a molecular laboratory is being realised. From the assay side of things, there are a plethora of newer chemistries for performing the PCR, although in reality they must compete with what is essentially a very easy to use and, perhaps more importantly, established format.”

Pfaffl: “The most important thing for the majority of researchers is the throughput. Therefore, all researchers are going for high throughput applications and want to perform lots of qPCR reactions in parallel on mRNA and on the microRNA level. The researchers want to have it all done in the minimum time, using low reaction volumes. That’s fine – but who cares about the validity of the produced results? Who has the capacity to check all the performed reactions?

Therefore, a future focus should be put on post-qPCR data analysis. First, check the integrity of your qPCR reaction as such. How was the performance of my PCR – low background, steep increase and high plateau? Then go for a reliable and reproducible background correction and threshold setting to get a reliable Cq value. Or better, use the so-called user independent Cq algorithms, like ‘second derivative maximum methods’ (Roche Applied Science), or ‘calqplex’ (Eppendorf ) or the ‘comparative quantification’ (Qiagen). They ensure you the best Cq data quality.

A further bottleneck is the proper data normalisation in expression profiling experiments. Still today – eight years after the key papers were published (e.g. Genorm, Normfinder or Bestkeeper) – scientists have severe problems in doing a proper and reliable normalisation. They should strictly follow the MIQE guidelines published last year by us (SA Bustin et al., Clin Chem, 2009) to increase validity and reliability of qRT-PCR results.”

Nour: “Quantitative PCR (qPCR) has revolutionised the way pharmaceutical laboratories discover biomarkers. In general, qPCR diagnosis is completed in an hour or even less (15 minutes in some specific conditions), which is considerably faster than conventional PCR and other detection methods (blotting, ELISA). When I started using qPCR for pathogen detection 10 years ago, we had to choose between two or three qPCR systems and reagent suppliers. Nowadays, more than 30 suppliers can provide qPCR solutions i.e. highly sophisticated reagents, machines with LED detection technologies and specialised bioinformatics software, which is proof of the exponential use of qPCR. The combination of improved sensitivity and specificity, low contamination risk, user-friendly software (Microsoft-based), ease of performance and speed has made qPCR technology the perfect alternative to conventional culture-based or immunoassay-based testing methods used for example in the clinical labora – tories for diagnostics tests.”

3. Which areas of drug discovery will benefit most from further qPCR research?

Pfaffl: “I see a big future in circulating serum or plasma derived microRNAs as diagnostic biomarkers and as well as potential therapeutic applications. The microRNA expression pattern in com – bination with meaningful analysis software tool (Genex, MultiD Sweden) and the appropriate algorithm (Principal Component Analysis, PCA) will accelerate validated biomarker discovery. The microRNAs regulate the gene expression, modulate the expression pattern and play an essential role in various cellular processes. Therefore, microRNAs are important for cell growth, apoptosis, proliferation and differentiation processes. I see a big future in the therapeutic applications of microRNA (or their complementary adaptamers) by controlling the transcriptome and proteome.”

Huggett: “Possibly one of the most powerful benefits of drug discovery will be from monitoring the impact of possible drugs on disease by measuring biomarkers, a fact that is increasingly possible with the development dynamic array PCR. Medicine will also further benefit as more diagnostic and prognostic tests are developed alongside the already established qPCR approaches. The improved precision offered by the digital PCR format opens the possibility of far finer measurements. The impact digital PCR could have on foetal aneuploidy analysis or early tumour detection need to be defined, but this approach offers exciting potential.”

4. Do you feel there are any areas of qPCR that are being held back and what changes do you think could unlock them?

Huggett: “Possibly one of the main areas where qPCR is being held back is by the lack of method – ological information present in published data. This makes external data interpretation and comparison very difficult and undoubtedly leads to data ambiguity and disagreement. Many problems also occur due to the fact that qPCR provides very precise results that can easily result in statistically significant, but biased findings; these can lead to an inaccurate estimation of the magnitude of a finding or, worse, a completely incorrect finding. This fact and its associated problem have probably had the most impact when qPCR is combined with the reverse transcription reaction to measure RNA gene expression. The MIQE guidelines were specifically developed to address this problem and will hopefully go some way to unlocking these issues.”

Nour: “As we mentioned before, qPCR has no equivalent in proteomics and this significantly decreases the use of qPCR in proteomics. Although qPCR is becoming a default technique in genomics and transcriptomics, applied to proteomics, qPCR remains in the developing phase. Recently, Ulf Landegren from Sweden invented a qPCR based assay for protein detection and identification. The proximity ligation assay (PLA) consists of specific affinity binders such as antibodies and/or DNAaptamers are conjugated with oligonucleotides having free 3’ or 5’ ends, creating proximity probes. PLA provides the specificity of an immunoassay with the sensitivity and manageability of PCR. Recently, multiplex PLA was used to identify and to validate sets of putative disease biomarkers relevant to cancer. Commercialising such techniques allows the introduction of qPCR in laboratories dealing with proteins and might cause a surge in qPCR market worldwide.”

Pfaffl: “No comment!”

5. Where do you see the qPCR industry in five years time?

Pfaffl: “This depends heavily on how the next generation sequencing and hybridisation array technologies develop, in terms of run and consumable prices, validity and reproducibility. A problem in these technologies will always be the limitation of biological material and therefore the available RNA for diagnostics, e.g. in laser micro-dissected tissue, tissue biopsies or single-cells. Of course, we can perform a pre-amplification step, but we know about the given problem to introduce an error and generate biased expression results. There I still see quantitative RT-PCR as the affordable ‘gold standard’ and as the market leader for the classical molecular biology lab worldwide.”

Nour: “Despite its youth, qPCR is widely applicable. Fields of applications are many and extremely diverse: molecular diagnostics, drug discovery, pre-clinical and clinical studies, pharmacology, toxicology, veterinary diagnostics, phytopath – ology and food quality. We have even used qPCR in the car industry by detecting microbes in the metal working fluids. Although the expansion of this technique seems to be unstoppable, many pitfalls are still to be considered:

i) The cost for setting up qPCR systems and for carrying out the experiments is still high for small laboratories and laboratories in the developing countries

ii) Miniaturisation even with the development of microfluidic systems footprints (HxWxD) of qPCR machines are high with the smallest footprint of Cepheid’s Smartcycler® (31x31x25 in centimetres)

iii) Sample preparation quality affects the quality of the qPCR experiments. IT specialist taught me a quote from their field; “garbage in, garbage out.”

We cannot expect much from poor quality samples. My ideal future for qPCR would be a cheap, matchbox size system that can directly detect pathogens from one or two drops of sample (blood).”

Huggett: “This is likely to be dominated with smaller volume, higher throughput, with point of care (or near patient care) being an important driver of the clinical diagnosis field. Isothermal methods like loop mediated amplification (LAMP) and recombinase polymerase reaction (RPA) also have the potential to considerably simplify instrumentation, reducing costs and speeding up reaction times, so these types of approaches are also likely to become more common. The industry is also likely to focus on more sensitive methods that do not require enzymatic amplification; how far this will come in the next five years remains to be seen, but the potential of techniques like third generation sequencing offers much. It is likely that in 50 years time, scientists will look back fondly on the PCR reaction as a historically fundamental yet quaint method where we, their predecessors, had to generate a billion copies of DNA before we could visualise anything.”

6. Is there anything else that you would like to add which you think would be relevant to our readers?

Nour: “qPCR techniques are developed in European countries as well as the USA. Not much is done in developing countries where the laboratories are in need of state-of-the-art techniques and researchers often have less training. In a recent paper published in Nature, genetics scientists with expertise in global health issues identified the top ten biotechnologies for improving health in developing countries. Second and third place were for techniques related to drug and vaccines development. We think the time has come for a technology transfer between the developed and the developing countries, especially to the countries where pathogens diagnostics is vital. qPCR can have a positive impact, if well used, in preventing diseases and health problems.”

Pfaffl: “In times of high throughput and pre-optimised assays, a validation of the entire workflow, especially in the pre-PCR steps, is becoming more and more important. From my varied experience carrying out expression profiling in multiple species and various tissues, the sampling and RNA fixation is one of the most potent steps to minimise variability and push reproducibility. Therefore, we strongly recommend following the MIQE guidelines to get the maximum out of your experimental trials.”

About the authors

Afif M Abdel Nour
Afif Abdel Nour has a PhD in Nutrigenomics and specialises in applied techniques in Molecular Biotechnology. He completed his Masters in Molecular Microbiology at Pasteur Institut Lille. Currently, he is working as a Biotechnology lecturer and Molecular Biotechnology consultant at the Institute Polytechnique LaSalle Beauvais, France and in several countries in the Middle East. He is helping set up a real-time PCR facility for routine diagnostics in these countries.

Contact the author: [email protected]

Michael Pfaffl
Michael Pfaffl is a reader in physiology and recognised teacher of the University of Munich. Michael has diplomas in Agriculture, Livestock Sciences and Biotechnology. Michael has over 100 original papers in international peer refereed journals and over 25 reviews and book chapters.

Jim Huggett
Jim Huggett obtained his PhD from Cardiff University investigating gene expression in bone disease. He moved to the Centre for Infectious Diseases at UCL, where his principle interests focused on molecular analysis of respiratory tract infections. He has recently moved to LGC to lead the diagnostics research for the Molecular and Cell Biology team. He is investigating the latest technologies for diagnosing a range of disease types including infection and cancer as well as foetal analysis. Jim’s research philosophy is driven by the desire to ensure correct findings and frequently results in challenging established dogma when appropriate. His publication record reflects this and he has lectured on this subject, as an invited speaker, at a numerous conferences and workshops.