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Developed in the mid 1990s for the analysis and quantification of nucleic acids, real-time PCR is a molecular biological technique gaining rapidly in popularity. It is based on the technique of the polymerase chain reaction (PCR) that was first envisioned by Kary Mullis almost 20 years ago, during a moonlit drive through the redwood hills of California (1). The technology of PCR (2) has become one of the most influential discoveries of the molecular biology revolution and one for which Mullis received the Nobel Prize in 1993. Because of the impact of PCR and the thermostable Taq DNA polymerase (the enzyme responsible for the PCR revolution), the pair was named as the first “Molecule of the Year” by Science in 1989 (3). In many ways, the recent development of real-time PCR seems set to change the general use of PCR. etc.

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Quantitative real-time RT-PCR – a perspective
S A Bustin, V Benes, T Nolan and M W Pfaffl

 

Bustin SA, Mueller R.
Mol Aspects Med. 2006 27(2-3):192-223

Institute of Cell and Molecular Science, Barts and the London, Queen Mary's
School of Medicine and Dentistry, University of London, UK.

Molecular diagnostics offers the promise of accurately matching patient with treatment, and a resultant significant effect on improved disease outcome. More specifically, the real-time reverse transcription polymerase chain reaction (qRT-PCR), with its combination of conceptual simplicity and technical utility, has the potential to become a valuable analytical tool for the detection of mRNA targets from tissue biopsies and body fluids. Its potential is particularly promising in cancer patients, both as a prognostic assay and for monitoring response to therapy. Colorectal cancer provides an instructive paradigm for this potential as well as the problems associated with its use as a clinical assay. Currently, histopathological staging, which provides a static description of the anatomical extent of tumour spread within a surgical specimen, defines patient prognosis. The detection of lymph node (LN) metastasis constitutes the most important prognostic factor in colorectal cancer and as the primary indicator of systemic disease spread, LN status determines the choice of postoperative adjuvant chemotherapy. However, its limitations are emphasised by the considerable prognostic heterogeneity of patients within a given tumour stage: not all patients with LN-negative cancers are cured and not all patients with LN-positive tumours die from their disease. This has resulted in a search for more accurate staging protocols and has seen the introduction of the concept of "molecular staging", the incorporation of molecular parameters into clinical tumour staging. Quantification of disease-associated mRNA is one such parameter that utilises the qRT-PCR assay's potential for generating quantitative results. These are not only more informative than qualitative data, but contribute to assay standardisation and quality management. This review provides an assessment of the practical value to the clinician of RT-PCR-based molecular diagnostics. It points out reasons for the many contradictory results encountered in the literature and concludes that there is an urgent need for standardisation at every level, starting with pre-assay sample acquisition and template preparation, assay protocols and post-assay analysis.

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Faster quantitative real-time PCR protocols may lose sensitivity and show increased variability.

Hilscher C, Vahrson W, Dittmer DP.
Nucleic Acids Res. 2005 Nov 27;33(21):e182.
 
Department of Microbiology and Immunology and Lineberger Comprehensive Cancer
Center, The University of North Carolina at Chapel Hill, NC, USA.

Quantitative real-time PCR has become the method of choice for measuring mRNA transcription. Recently, fast PCR protocols have been developed as a means to increase assay throughput. Yet it is unclear whether more rapid cycling conditions preserve the original assay performance characteristics. We compared 16 primer sets directed against Epstein-Barr virus (EBV) mRNAs using universal and fast PCR cycling conditions. These primers are of clinical relevance, since they can be used to monitor viral oncogene and drug-resistance gene expression in transplant patients and EBV-associated cancers. While none of the primers failed under fast PCR conditions, the fast PCR protocols performed worse than universal cycling conditions. Fast PCR was associated with a loss of sensitivity as well as higher variability, but not with a loss of specificity or with a higher false positive rate.

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Comparison of in vitro and in vivo reference genes for internal standardization of real-time PCR data.

Gilsbach R, Kouta M, Bonisch H, Bruss M.
Biotechniques. 2006 40(2): 173-177.

Institute of Pharmacology and Toxicology, University of Bonn, Bonn, Germany.

Real-time PCR is a powerful technique for gene expression studies, which have become increasingly important in a large number of clinical and scientific fields. The significance of the obtained results strongly depends on the normalization of the data to compensate for differences between the samples. The most widely used approach is to use endogenous reference genes (housekeeping genes) as internal standards. This approach is controversially discussed in the literature because none of the reference genes is stably expressed throughout all biological samples. Therefore, candidate reference genes have to be validated for each experimental condition. In our studies, we introduced and evaluated an in vitro synthesized reference cRNA for internal standardization of relative messenger RNA (mRNA) expression patterns. This reference, consisting of the in vitro transcribed coding sequence of aequorin, a jellyfish protein, was incorporated in the extracted RNA. The experimental significance of this approach was representatively tested for the expression of the neurotrophin-3 mRNA in distinct regions of mouse brains. A comparison to three stably expressed reference genes [beta-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and hypoxanthine phosphoribosyl-transferase 1 (HPRT1)] gave evidence that the spiking of template RNA with in vitro transcribed cRNA is a valuable tool for internal standardization of real-time PCR experiments.

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Real-Time PCR     and    Real-Time Reverse Transcription PCR

two interesting reviews  by  Stephen A. Bustin, University of London, London, U.K.
Encyclopedia of Diagnostic Genomics and Proteomics

Real-Time PCR The real-time polymerase chain reaction (PCR) uses fluorescent reporter molecules to monitor the production of amplification products during each cycle of the PCR reaction. This combines the DNA amplification and detection steps into one homogeneous assay and obviates the need for gel electrophoresis to detect amplification products. Appropriate data analysis and/or use of apposite chemistries also eliminates the need for Southern blotting or DNA sequencing for amplicon identification. Its simplicity, specificity, and sensitivity, together with its potential for high throughput and the ongoing introduction of new chemistries, more reliable instrumentation, and improved protocols, has made realtime PCR the benchmark technology for the detection of DNA.

Real-Time Reverse Transcription PCR Real-time, fluorescence-based reverse transcription polymerase chain reaction (RT-PCR) has been transformed from an experimental technology into a mainstream scientific tool for the detection of RNA. This is because of several factors: 1) it is a homogeneous assay, which eliminates the requirement for post-PCR processing; 2) it has a wide dynamic range; 3) there is little interassay variation; and 4) it realizes the inherent quantitative capacity of PCR-based assays, making it a quantitative rather than a qualitative, assay. These properties match the evident requirement in molecular medicine for quantitative data (e.g., for measuring viral load, monitoring of occult disease in cancer, or examining the genetic basis for individual variation in response to therapeutics through pharmacogenomics).

The power of real-time PCR.
Valasek MA, Repa JJ.

Adv Physiol Educ. 2005 Sep;29(3): 151-159.

Department of Physiology Touchstone Center for Diabetes Research, University of
Texas Southwestern Medical Center, Dallas, Texas.

In recent years, real-time polymerase chain reaction (PCR) has emerged as a robust and widely used methodology for biological investigation because it can detect and quantify very small amounts of specific nucleic acid sequences. As a research tool, a major application of this technology is the rapid and accurate assessment of changes in gene expression as a result of physiology, pathophysiology, or development. This method can be applied to model systems to measure responses to experimental stimuli and to gain insight into potential changes in protein level and function. Thus physiology can be correlated with molecular events to gain a better understanding of biological processes. For clinical molecular diagnostics, real-time PCR can be used to measure viral or bacterial loads or evaluate cancer status. Here, we discuss the basic concepts, chemistries, and instrumentation of real-time PCR and include present applications and future perspectives for this technology in biomedical sciences and in life science education.

Michael W. Pfaffl,   Physiology, Department of Animal Science,
Center of Life and Food Sciences,  Weihenstephaner Berg 3, 85354 Freising, Germany

Molecular technologies are currently evolving rapidly in agricultural and veterinary sciences. This results in an immense progress in the accumulation of new data potentially useful for molecular diagnostics in farm animal physiology, immunology, diseases and new breeding strategies. While we are still at the “very beginning” of understanding genomics, transcriptomics and proteomics in relation to animal physiology, this development has dramatically changed our perspectives in research during the last decade. It can be foreseen, that the application of sophisticated rather than simple methods will be necessary for numerous diagnostic questions. One of this highly sophisticated methodologies is the quantitative assessment of target nucleic acids, mostly performed as quantitative polymerase chain reaction (PCR) on DNA level or combined with reverse transcription PCR (RT-PCR) to investigate the transcriptome on RNA level. This review will introduce the state of the art in quantitative RT-PCR using real-time RT-PCR on the field of livestock molecular endocrinology and physiology.

Real-time RT-PCR:  Neue Ansätze zur exakten mRNA Quantifizierung
Michael W. Pfaffl (2004)
BioSpektrum 1/2004 (in German)

Die molekularen Technologien Genomics, Transcriptomics und Proteomics erobern immer mehr die klassischen Forschungsgebiete der Biowissenschaften. Die enorme Flut an gewonnenen Daten und Ergebnissen ist von überproportionalem Nutzen in der molekularen Diagnostik und Physiologie sowie die „Functional Genomics“. Immer neue ausgeklügelte Methoden und Anwendungen sind daher nötig um komplexe physiologische Vorgänge zu beschreiben. Da wir uns erst an Anfang dieser molekularen Ära befinden, ist es notwendig diese Techniken zu optimieren und komplett zu verstehen. Eine dieser technisch ausgefeilten Methoden zur zuverlässigen und exakten Quantifizierung spezifischer mRNA, stellt die real-time RT-PCR dar. Dieser Artikel beschreibt im Wesentlichen die effizienzkorrigierte relative Quantifizierung, die Normalisierung der Expressionsergebnisse anhand eines nicht regulierten „Housekeeping Gens“, die Berechnung der real-time PCR Effizienz sowie die Verrechnung und statistische Auswertung der Expressionsergebnisse. Alle beschriebenen Themenkomplexe können im Detail auf der korrespondierenden Internetseite (http://www.gene-quantification.info) in internationalen publizierten Originalarbeiten nachgeschlagen werden.

Relative transcript quantification by Quantitative PCR:  Roughly right or precisely wrong ?
Rasmus Skern, Petter Frost and Frank Nilsen

Background: When estimating relative transcript abundances by quantitative real-time PCR (QPCR) we found that the results can vary dramatically depending on the method chosen for data analysis.
Results: Analyses of Q-PCR results from a salmon louse starvation experiment show that, even with apparently good raw data, different analytical approaches [1,2] may lead to opposing biological conclusions.
Conclusion: The results emphasise the importance of being cautious when analysing Q-PCR data and indicate that uncritical routine application of an analytical method will eventually result in incorrect conclusions. We do not know the extent of, or have a universal solution to this problem. However, we strongly recommend caution when analysing Q-PCR results e.g. by using two or more analytical approaches to validate conclusions. In our view a common effort should be made to standardise methods for analysis and validation of Q-PCR results.

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Real-time RT-PCR normalisation;  strategies and considerations
J Huggett, K Dheda, S Bustin and A Zumla

Real-time RT-PCR has become a common technique, no longer limited to specialist core facilities. It is in many cases the only method for measuring mRNA levels of vivo low copy number targets of interest for which alternative assays either do not exist or lack the required sensitivity. Benefits of this procedure over conventional methods for measuring RNA include its sensitivity, large dynamic range, the potential for high throughout as well as accurate quantification. To achieve this, however, appropriate normalisation strategies are required to control for experimental error introduced during the multistage process required to extract and process the RNA. There are many strategies that can be chosen; these include normalisation to sample size, total RNA and the popular practice of measuring an internal reference or housekeeping gene. However, these methods are frequently applied without appropriate validation. In this review we discuss the relative merits of different normalisation strategies and suggest a method of validation that will enable the measurement of biologically meaningful results.

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MICROARRAY TECHNOLOGIES
Validation of oligonucleotide microarray data using microfluidic low-density arrays:
a new statistical method to normalize real-time RT-PCR data.
Lynne V. Abruzzo et al. BioTechniques 38:785-792 (May 2005)

Profiling studies using microarrays to measure messenger RNA (mRNA) expression frequently identify long lists of differentially expressed genes. Differential expression is often validated using real-time reverse transcription PCR (RT-PCR) assays. In conven-tional real-time RT-PCR assays, expression is normalized to a control, or housekeeping gene. However, no single housekeeping gene can be used for all studies. We used TaqMan® Low-Density Arrays, a medium-throughput method for real-time RT-PCR using microfluidics to simultaneously assay the expression of 96 genes in nine samples of chronic lymphocytic leukemia (CLL). We devel-oped a novel statistical method, based on linear mixed-effects models, to analyze the data. This method automatically identifies the genes whose expression does not vary significantly over the samples, allowing them to be used to normalize the remaining genes. We compared the normalized real-time RT-PCR values with results obtained from Affymetrix Hu133A GeneChip® oligonucleotide microarrays. We found that real-time RT-PCR using TaqMan Low-Density Arrays yielded reproducible measurements over seven or-ders of magnitude. Our model identified numerous genes that were expressed at nearly constant levels, including the housekeeping genes PGK1, GAPD, GUSB, TFRC, and 18S rRNA. After normalizing to the geometric mean of the unvarying genes, the correla-tion between real-time RT-PCR and microarrays was high for genes that were moderately expressed and varied across samples.

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Linear-After-The-Exponential (LATE)-PCR: Primer design criteria for high yields of specific singlestranded
DNA and improved real-time detection.
Kenneth E. Pierce, J. Aquiles Sanchez, John E. Rice, and Lawrence J. Wangh

Traditional asymmetric PCR uses conventional PCR primers at unequal concentrations to generate single-stranded DNA. This method, however, is difficult to optimize, often inefficient, and tends to promote nonspecific amplification. An alternative approach, Linear-After-The-Exponential (LATE)-PCR, solves these problems by using primer pairs deliberately designed for use at unequal concentrations. The present report systematically examines the primer design parameters that affect the exponential and linear phases of LATE-PCR amplification. In particular, we investigated how altering the concentration-adjusted melting temperature (Tm) of the limiting primer (Tm L) relative to that of the excess primer (Tm X) affects both amplification efficiency and specificity during the exponential phase of LATE-PCR. The highest reaction efficiency and specificity were observed when Tm L
Tm X>5°C. We also investigated how altering Tm X relative to the higher Tm of the double-stranded amplicon (Tm A) affects the rate and extent of linear amplification. Excess primers with Tm X closer to Tm A yielded higher rates of linear amplification and stronger signals from a hybridization probe. These design criteria maximize the yield of specific single-stranded DNA products and make LATE-PCR more robust and easier to implement. The conclusions were validated by using primer pairs that amplify sequences within the cystic fibrosis transmembrane regulator (CFTR) gene, mutations of which are responsible for cystic fibrosis.

Real-Time PCR Technology for Cancer Diagnostics
Philip S. Bernard and Carl T. Wittwer
Clinical Chemistry 48: 8  1178–1185 (2002)