Real-time PCR in the microbiology laboratory
I. M. Mackay

Clinical Virology Research Unit, Sir Albert Sakzewski Virus Research Centre and Department of
Paediatrics, Royal Children’s Hospital, Brisbane, Queensland, Australia
Clin Microbiol Infect 2004; 10: 190–212

Use of PCR in the field of molecular diagnostics has increased to the point where it is now accepted as the standard method for detecting nucleic acids from a number of sample and microbial types. However, conventional PCR was already an essential tool in the research laboratory. Real-time PCR has catalysed wider acceptance of PCR because it is more rapid, sensitive and reproducible, while the risk of carryover contamination is minimised. There is an increasing number of chemistries which are used to detect PCR products as they accumulate within a closed reaction vessel during real-time PCR. These include the non-specific DNA-binding fluorophores and the specific, fluorophore-labelled oligonucleotide probes, some of which will be discussed in detail. It is not only the technology that has changed with the introduction of real-time PCR. Accompanying changes have occurred in the traditional terminology of PCR, and these changes will be highlighted as they occur. Factors that have restricted the development of multiplex real-time PCR, as well as the role of real-time PCR in the quantitation and genotyping of the microbial causes of infectious disease, will also be discussed. Because the amplification hardware and the fluorogenic detection chemistries have evolved rapidly, this review aims to update the scientist on the current state of the art. Additionally, the advantages, limitations and general background of real-time PCR technology will be reviewed in the context of the microbiology laboratory.

An overview of real-time quantitative PCR:  applications to quantify cytokine gene expression.

Giulietti A, Overbergh L, Valckx D, Decallonne B, Bouillon R, Mathieu C. (2001)
Methods  2001 25(4): 386-401

Laboratory for Experimental Medicine and Endocrinology (LEGENDO), Catholic
University of Leuven, Herestraat 49, 3000 Leuven, Belgium.

The analysis of cytokine profiles helps to clarify functional properties of immune cells, both for research and for clinical diagnosis. The real-time reverse transcription polymerase chain reaction (RT-PCR) is becoming widely used to quantify cytokines from cells, body fluids, tissues, or tissue biopsies. Being a very powerful and sensitive method it can be used to quantify mRNA expression levels of cytokines, which are often very low in the tissues under investigation. The method allows for the direct detection of PCR product duringthe exponential phase of the reaction, combining amplification and detection in one single step. In this review we discuss the principle of real-time RT-PCR, the different methodologies and chemistries available, the assets, and some of the pitfalls. With the TaqMan chemistry and the 7700 Sequence Detection System (Applied Biosystems), validation for a large panel of murine and human cytokinesnd other factors playing a role in the immune system is discussed in detail. In summary, the real-time RT-PCR technique is very accurate and sensitive, allows a high throughput, and can be performed on very small samples; therefore it is the method of choice for quantification of cytokine profiles in immune cells or inflamed tissues.

Quantitative reverse transcription-polymerase chain reaction to 
study mRNA decay: comparison of endpoint and real-time methods.

Schmittgen TD, Zakrajsek BA, Mills AG, Gorn V, Singer MJ, Reed MW.
Anal Biochem  2000 285(2):194-204

Department of Pharmaceutical Sciences, College of Pharmacy, Washington State
University, Pullman, Washington 99164-6534, USA.

Four quantitative reverse transcription-PCR (RT-PCR) methods were compared to evaluate the time course of mRNA formation and decay. Mouse fibroblasts (NIH 3T3) transfected with the human beta-globin open reading frame/c-myc 3'-untranslated region chimeric gene under control of the c-fos promoter (fos-glo-myc) were used for serum-inducible transcription. The amount of fos-glo-myc mRNA, relative to beta-actin, was measured by quantitative, RT-PCR at various times following the addition of serum to serum-starved fibroblasts transfected with the chimeric gene. Both endpoint (band densitometry and probe hybridization) and real-time (SYBR green and TaqMan) PCR methods were used to assay the identical cDNA. The real-time methods produced a 4- to 5-log dynamic range of amplification, while the dynamic range of the endpoint assays was 1-log. The real-time and probe hybridization assays produced a comparable level of sensitivity that was considerably greater than band densitometry. The coefficient of variation from 22 replicate PCR reactions was 14.2 and 24.0% for the SYBR green and TaqMan detection, respectively, and 44.9 and 45.1% for the band densitometry and probe hybridization assays, respectively. The rank order for the values of r(2) obtained from the linear regression of the first-ordermRNA decay plots was SYBR green > TaqMan > probe hybridization > band densitometry. Real-time PCR is more precise and displays a greater dynamic range than endpoint PCR. Among the real-time methods, SYBR green and TaqMan assays produced comparable dynamic range and sensitivity while SYBR green detection was more precise and produced a more linear decay plot than TaqMan detection.

Real-time PCR in virology

Mackay IM, Arden KE, Nitsche A.

Nucleic Acids Res. 2002 30(6):1292-305. Review.

          Clinical Virology Research Unit, Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital, Brisbane, Australia.

The use of the polymerase chain reaction (PCR) in molecular diagnostics has increased to the point where it is now accepted as the gold standard for detecting nucleic acids from a number of origins and it has become an essential tool in the research laboratory. Real-time PCR has engendered wider acceptance of the PCR due to its improved rapidity, sensitivity, reproducibility and the reduced risk of carry-over contamination. There are currently five main chemistries used for the detection of PCR product during real-time PCR. These are the DNA binding fluorophores, the 5' endonuclease, adjacent linear and hairpin oligoprobes and the self-fluorescing amplicons, which are described in detail. We also discuss factors that have restricted the development of multiplex real-time PCR aswell as the role of real-time PCR in quantitating nucleic acids. Both amplification hardware and the fluorogenic detection chemistries have evolved rapidly as the understanding of real-time PCR has developed and this review aims to update the scientist on the current state of the art. We describe the background, advantages and limitations of real-time PCR and we review the literature as it applies to virus detection in the routine and research laboratory in order to focus on one of the many areas in which the application of real-time PCR has provided significant methodological benefits and improved patient outcomes. However, the technology discussed has been applied to other areas of microbiology as well as studies of gene expression and genetic disease.

Quantitative RT-PCR:  pitfalls and potential

Freeman, WM, Walker, SJ, and Vrana, KE (1999) 
 Biotechniques 26, 112-122.

Reverse transcription PCR (RT-PCR) represents a sensitive and powerful tool for analyzing RNA. While it has tremendous potential for quantitative applications, a comprehensive knowledge of its technical aspects is required. Successful quantitative RT-PCR involves correction for experimental variations in individual RT and PCR efficiencies. This review addresses the mathematics of RT-PCR, choice of RNA standards (internal vs. external) and quantification strategies (competitive, noncompetitive and kinetic [real-time] amplification). Finally, the discussion turns to practical considerations in experimental design. It is hoped that this review will be appropriate for those undertaking these experiments for the first time or wishing to improve (or validate) a technique in what is frequently a confusing and contradictory field.

Quantification using real-time PCR technology:  Applications and limitations

Klein D. (2002)
Trends Mol Med  2002 (6): 257-260 

The introduction of real-time PCR technology has significantly improved and simplified the quantification of nucleic acids, and this technology has become an invaluable tool for many scientists working in different disciplines. Especially in the field of molecular diagnostics, real-time PCR-based assays have gained favour in the recent past. However, the wide use of real-time PCR methods has also highlighted some of the critical points and limitations of these assays. These aspects must be considered to increase the reliability of the obtained data.

Gene quantification using real-time quantitative PCR:  An emerging technology hits the mainstream

Ginzinger DG.

Exp Hematol  2002 30(6): 503-512
Genome Analysis Core Facility, Comprehensive Cancer Center, School of Medicine,
University of California at San Francisco, San Francisco, Calif., USA

The recent flood of reports using real-time Q-PCR testifies to the transformation of this technology from an experimental tool into the scientific mainstream. Many of the applications of real-time Q-PCR include measuring mRNA expression levels, DNA copy number, transgene copy number and expression analysis, allelic discrimination, and measuring viral titers. The range of applications of real-time Q-PCR is immense and has been fueled in part by the proliferation of lower-cost instrumentation and reagents. Successful application of real-time Q-PCR is not trivial. However, this review will help guide the reader through the variables that can limit the usefulness of this technology. Careful consideration of the assay design, template preparation, and analytical methods are essential for accurate gene quantification.

PCR Papers from HorizonPress 2008

An Introduction to Real-Time PCR
N.A. Saunders

The development of instruments that allowed real-time monitoring of fluorescence within PCR reaction vessels was a very significant advance. The technology is very flexible and many alternative instruments and fluorescent probe systems have been developed and are currently available. Real-time PCR assays can be completed very rapidly since no manipulations are required post-amplification. Identification of the amplification products by probe detection in real-time is highly accurate compared with size analysis on gels.

Real-Time PCR Platforms
M.J. Logan and K.J. Edwards

Real-time PCR continues to have a major impact across many disciplines of the biological sciences and this has been a driver to develop and improve existing instruments. From the first two commercial platforms introduced in the mid 1990s, there is now a choice in excess of a dozen instruments, which continues to increase. Advances include faster thermocycling times, higher throughput, flexibility, expanded optical systems, increased multiplexing and more user-friendly software.

Performing Real-Time PCR
K.J. Edwards

Optimisation of the reagents used to perform PCR is critical for reliable and reproducible results. As with any PCR initial time spent on optimisation of a real-time assay will be beneficial in the long run. Specificity, sensitivity, efficiency and reproducibility are the important criteria to consider when optimising an assay and these can be altered by changes in the primer concentration, probe concentration, cycling conditions and buffer composition. An optimised real-time PCR assay will display no test-to-test variation in the crossing threshold or crossing point and only minimal variation in the amount of fluorescence.

Internal and External Controls for Reagent Validation
M.A. Lee, D.L. Leslie and D.J. Squirrell

PCR applications that require a high confidence in the result should be designed to control for the occurrence of false negatives. False negatives can occur from inhibition of one or more of the reaction components by a range of factors. While an external, or batch control is often used, the ideal control is one that is included in the reaction cocktail in a multiplex format. Early approaches used different sized amplicons combined with end-point analysis. Fluorescent homogenous real-time PCR methods have a number of advantages for implementing internal controls.

Quantitative Real-Time PCR
N.A. Saunders

Unlike classical end-point analysis PCR, real-time PCR provides the data required for quantification of the target nucleic acid. The results can be expressed in absolute terms by reference to external quantified standards or in relative terms compared to another target sequence present within the sample. Absolute quantification requires that the efficiency of the amplification reaction is the same in all samples and in the external quantified standards. Consequently, it is important that the efficiency of the PCR does not vary greatly due to minor differences between samples. Careful optimisation of the PCR conditions is therefore required. The use of probes in quantitative real-time PCR improves its performance and a range of suitable systems is now available.

Analysis of mRNA Expression by Real-Time PCR
S.A. Bustin and T. Nolan

The last few years have seen the transformation of the fluorescence-based real-time reverse transcription polymerase chain reaction (RT-PCR) from an experimental tool into a mainstream scientific technology. Assays are simple to perform, capable of high throughput, and combine high sensitivity with exquisite specificity. The technology is evolving rapidly with the introduction of new enzymes, chemistries and instrumentation and has become the "Gold Standard" for a huge range of applications in basic research, molecular medicine, and biotechnology.