from Vijay J. Gadkar and Martin Filion writing in Quantitative Real-time PCR in Applied Microbiology:
Environmental matrices are highly diverse in their composition and range from simple (e.g. water) to highly complex (e.g. organic soils/biosolids). Analysis of microbial gene expression from such substrates is done for variety of purposes which could range from bio-surveillance to elucidation of biological function of a target microbe. Quantitative real-time PCR (RT-qPCR) has become a technique of choice for studying such bio-processes, due to its unique ability to both detect and quantify a target transcript in real-time. Challenges in extracting inhibitor-free, structurally intact RNA, amenable for a sensitive technique like RT-qPCR, has however proved to be a major impediment in our ability to rigorously implement this highly versatile technology. Despite these "substrate defined" limitations, many attempts have been made to implement the RT-qPCR technology. Efforts like these have given us invaluable insight into the expression status of a particular transcript and hence, the biological functioning of the microbe, specifically under natural in situ conditions. As a result, it has enhanced our understanding of the role and diversity of many microbial populations which, previously was not possible using conventional molecular approaches. In this chapter, we have sought to summarize such technical problems faced by molecular environmental microbiologist and solutions developed to mitigate those challenges.
Further reading: Quantitative Real-time PCR in Applied Microbiology Related publications
from Dan Tulpan, Michelle Davey and Mark Laflamme writing in Quantitative Real-time PCR in Applied Microbiology:
The ability of DNA microarray technology to identify and quantify microbial entities and genes of interest in various environments, such as soil, water, air, compost, and blood, propelled biological, environmental and clinical research into the post-genomic era. Nevertheless, as it is valid for any new technology, errors may occur at different stages along the experimental process. Three sources of errors associated with DNA microarray utilization have been identified by Taniguchi et al. (2001), namely: (i) the microarray fabrication, (ii) the microarray experiment, and (iii) the interpretation of results (data analysis). Validation strategies are typically required to alleviate and eventually repair the undesired errors that may arise in a microarray experiment. One of the validation techniques widely accepted and used worldwide is the quantitative Reverse Transcriptase Polymerase Chain Reaction (RT-qPCR). This chapter will provide succinct introductions to microarray technologies applied to microbial research and fundamental notions regarding RT-qPCR and its use to validate microarray results. A discussion including advantages and disadvantages of microbial microarray validation using RT-qPCR will be presented and current and future trends and research directions will be summarized towards the end of the chapter.
Further reading: Quantitative Real-time PCR in Applied Microbiology Related publications
from Michael W. Pfaffl writing in Quantitative Real-time PCR in Applied Microbiology:
The present chapter describes the quantification strategies used in real-time RT-PCR (RT-qPCR), focusing on the main elements that are essential to fulfil the MIQE guidelines. The necessity of initial proper data adjustment and background correction is discussed to allow reliable quantification. The advantages and disadvantages of the absolute and relative quantification approaches are also described. In conjunction with relative quantification, the importance of an amplification efficiency correction is shown, and software tools that are available to calculate relative expression changes are presented.
Further reading: Quantitative Real-time PCR in Applied Microbiology Related publications
from Lia C.R.S. Teixeira and Etienne Yergeau writing in Quantitative Real-time PCR in Applied Microbiology:
Quantitative polymerase chain reaction (qPCR) represents an effective method to quantify genes or transcripts within environmental samples. For that reason, qPCR has been widely used to characterize the functional patterns of complex microbial communities. In this chapter we summarize some recent applications of different qPCR approaches targeting functional genes encoding key enzymes in the N-, C- and S-cycles and also functional genes related to antibiotic resistance. We also point out some limitation of qPCR approaches. The ongoing development of new molecular techniques, like metagenomics, will have positive impacts on the specificity and the coverage of qPCR assays, since the availability of more sequence data will help to improve the design of primers targeting functional genes.
Further reading: Quantitative Real-time PCR in Applied Microbiology Related publications
from Claudia Goyer and Catherine E. Dandie writing in Quantitative Real-time PCR in Applied Microbiology:
Development of quantitative PCR (qPCR) has facilitated major advances in assessment of microbial community abundances in complex environmental samples including water, soil, sediments, compost and manure and in our understanding of factors influencing community sizes in situ. qPCR has increasingly been used in environmental studies due to its sensitivity, ease of use, and the capacity to run large numbers of samples. However, qPCR has some limitations, which are specifically caused by the nature of environmental samples, including the variability in microorganism distribution, the efficiency of DNA recovery and purification, and the amount and type of PCR inhibitors co-extracted with the target nucleic acids. The heterogeneity of the templates amplified by qPCR can generate PCR biases and artifacts. Accuracy of the quantification of broad groups of microorganisms is influenced by the number of gene copies per genome of the selected marker. In this review, we will discuss the main experimental considerations for using qPCR in environmental studies, including the factors affecting key steps in the process of performing quantification of microorganisms in environmental samples. Although quantification of microorganisms is challenging, it is possible to reliably quantify microorganisms in complex environmental samples using qPCR. We will also briefly review the findings of studies which have used qPCR to quantify microorganisms from complex matrices.
Further reading: Quantitative Real-time PCR in Applied Microbiology Related publications
from Vijay J. Gadkar and Martin Filion writing in Quantitative Real-time PCR in Applied Microbiology:
Environmental matrices are highly diverse in their composition and range from simple (e.g. water) to highly complex (e.g. organic soils/biosolids). Analysis of microbial gene expression from such substrates is done for variety of purposes which could range from bio-surveillance to elucidation of biological function of a target microbe. Quantitative real-time PCR (RT-qPCR) has become a technique of choice for studying such bio-processes, due to its unique ability to both detect and quantify a target transcript in real-time. Challenges in extracting inhibitor-free, structurally intact RNA, amenable for a sensitive technique like RT-qPCR, has however proved to be a major impediment in our ability to rigorously implement this highly versatile technology. Despite these "substrate defined" limitations, many attempts have been made to implement the RT-qPCR technology. Efforts like these have given us invaluable insight into the expression status of a particular transcript and hence, the biological functioning of the microbe, specifically under natural in situ conditions. As a result, it has enhanced our understanding of the role and diversity of many microbial populations which, previously was not possible using conventional molecular approaches. In this chapter, we have sought to summarize such technical problems faced by molecular environmental microbiologist and solutions developed to mitigate those challenges.
Further reading: Quantitative Real-time PCR in Applied Microbiology
from Luca Cocolin and Kalliopi Rantsiou writing in Quantitative Real-time PCR in Applied Microbiology:
Since its first application in food microbiology in the late '90s, quantitative PCR (qPCR) has attracted the interest of researchers, working mainly in the field of food safety, but lately also of microbiologists studying spoilage and fermentation processes. In addition to the advantages that conventional PCR offers in microbiological testing, i.e. specificity, reduced time of analysis and detection of viable but not culturable cells, qPCR allows quantification of target populations. This aspect is particularly relevant for foodborne pathogens, for which specific microbiological criteria exist, but also for spoilage and technological important microorganisms, in order to follow their population kinetics in foods. Although advancements in food microbiology have been made from its application, qPCR has not yet been utilized to its full potential: the quantification step is only rarely carried out and qPCR is often used as an alternative of conventional PCR. In this chapter we will critically describe the application of qPCR in food microbiology based on the available literature, taking into account the specific problems and suggesting some possible solutions.
Further reading: Quantitative Real-time PCR in Applied Microbiology
from Mathilde H. Josefsen, Charlotta Löfström, Trine Hansen, Eyjólfur Reynisson and Jeffrey Hoorfar writing in Quantitative Real-time PCR in Applied Microbiology:
The polymerase chain reaction has revolutionized the world of scientific research and its broad application has caused a tremendous development of versatile PCR instruments and chemistries to fit its purpose. This chapter provides the reader with a general introduction to the basics of real-time PCR instrumentation, including the thermal and optical systems and the software. Performance parameters such as temperature uniformity, accuracy and ramp speed as well as reaction format, optical systems, calibration of dyes, software and comparison between different real-time PCR platforms will be discussed from a user perspective leading to an instrument selection guide. Differences between fluorescent DNA binding dyes and target-specific fluorescently labeled primers or probes for detection of amplicon accumulation will be discussed, along with the properties and applications of the most frequently applied chemistries. The fluorophores and quenchers used for primer and probe labeling and their compatibility will be presented, and finally the future challenges and trends within the field of qPCR instrumentation will be discussed.
Further reading: Quantitative Real-time PCR in Applied Microbiology Related publications
from Mikael Kubista, Vendula Rusnakova, David Svec, Björn Sjögreen and Ales Tichopad writing in Quantitative Real-time PCR in Applied Microbiology:
As the qPCR field advances, the design of experiments and the analysis of data is becoming more important and more challenging. Calculation of relative expression of a reporter gene to a reference gene in pairs of samples using the ΔΔCq method is no longer sufficient. Studies are now designed using multiple markers, nested levels, exploring or confirming the effect of multiple factors, occasionally in paired designs, etc. Proper handling of such data requires software that support the planning and design of experiments, and data analysis. Several software with these capacities are emerging. This chapter describes some of the features of one of the most powerful of those: GenEx from MultiD Analysis.
Further reading: Quantitative Real-time PCR in Applied Microbiology Related publications
from Vijay J. Gadkar and Martin Filion writing in Quantitative Real-time PCR in Applied Microbiology:
Real time-quantitative PCR (RT-qPCR) technology has revolutionized the detection landscape in every area of molecular biology. The fundamental basis of this technology has remained unchanged since its inception, however various modifications have enhanced the overall performance of this highly versatile technology. These improvements have ranged from changes in the individual components of the enzymatic reaction cocktail (polymerizing enzymes, reaction buffers, probes, etc.) to the detection system itself (instrumentation, software, etc). The RT-qPCR technology currently available to researchers is more sensitive, faster and affordable than when this technology was first introduced. In this chapter, we summarize the developments of the last few years in RT-qPCR technology and nucleic acid amplification.
Further reading: Quantitative Real-time PCR in Applied Microbiology Related publications
from Stephen A Bustin, Sara Zaccara and Tania Nolan writing in Quantitative Real-time PCR in Applied Microbiology:
The real-time fluorescence-based quantitative polymerase chain reaction (qPCR) has become the benchmark technology for the detection of nucleic acids in every area of microbiology, biomedical research, biotechnology and in forensic applications. Unlike conventional (legacy) PCR, which is a qualitative end-point assay, qPCR allows accurate quantification of amplified DNA in real time during the exponential phase of the reaction. The cost of instruments and reagents is well within reach of individual laboratories, assays are easy to perform, capable of high throughput and combine high sensitivity with reliable specificity. It is possible to achieve accurate and biologically meaningful quantification if meticulous attention is paid to the details of every step of the qPCR assay, starting with sample selection, acquisition and handling through assay design, validation and optimisation. The growing awareness of the need for standardisation, quality control and the significant problems associated with inadequate reporting of the assay has resulted in the publication of guidelines for minimum information for the publication of qPCR experiments (MIQE).
Further reading: Quantitative Real-time PCR in Applied Microbiology Related publications
from Gregory L. Shipley writing in PCR Troubleshooting and Optimization: The Essential Guide:
The MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines have been presented to serve as a practical guide for authors when publishing experimental data based on real-time qPCR. Each item is presented in tabular form as a checklist within the MIQE manuscript. However, this format has left little room for explanation of precisely what is expected from the items listed and no information on how one might go about assimilating the information requested. This chapter presents an expanded explanation of the guideline items with commentary on how those requirements might be met prior to publication.
Further reading: PCR Troubleshooting and Optimization: The Essential Guide
from Ian Kavanagh, Gerwyn Jones and Saima Naveed Nayab writing in PCR Troubleshooting and Optimization: The Essential Guide:
Whilst qPCR is a powerful technique, the results achieved using this method is valid only if the appropriate controls have been included in the experiment. Careful selection of controls and proper optimisation of qPCR conditions promise generation of highly specific, repeatable, reproducible and sensitive data. This chapter discusses the strategies for preparing both negative and positive controls for PCR, when they should be employed and how to interpret the information they provide. It also highlights the significance of standard curves for determining the initial starting amount of the target template and for assessing assay efficiency, precision, sensitivity, and dynamic range. It also provides guidance on how to prepare standards, interpret standard curve and troubleshoot inefficient qPCR reactions.
Further reading: PCR Troubleshooting and Optimization: The Essential Guide
from Martina Reiter and Michael W. Pfaffl writing in PCR Troubleshooting and Optimization: The Essential Guide:
PCR technology is based on a simple principle; an enzymatic reaction that increases the amount of nucleic acids initially present in a sample but this powerful method makes it possible to detect specific mRNA transcripts in any biological sample by the application of RT-PCR. The RT-PCR quantitative analysis workflow has several steps, each of which is crucial to the success of the experiment. It starts with a sampling step, followed by nucleic acid extraction and stabilization, cDNA synthesis and finally the qPCR where the mRNA quantification takes place. PCR itself is quite a stable reaction with reproducibility between 2-8% but the number and nature of the pre-PCR steps mean that there are many sources of experimental variance in the workflow. Reliable data can only be produced when the experimental variance is minimized, so the sources of variation must be identified and optimized for each step of each experiment. Typically, however, the pre-PCR steps are neglected and optimization is done for PCR reaction only. In this chapter the optimization of the whole RT-PCR workflow will be discussed and recommendations to reduce experimental variance and produce more reproducible and reliable results are put forward.
Further reading: PCR Troubleshooting and Optimization: The Essential Guide
from Sandrine Javorski-Miller and Ivan Delgado Orlic writing in PCR Troubleshooting and Optimization: The Essential Guide:
A paper from 2008 mentions that quantitative PCR is 25 years old (VanGuilder et al., 2008) but routine use of this technology has only taken off during the past 12 years. The first commercial Real-Time PCR instrument, the ABI Prism 7700, was introduced to researchers in 1996 by Applied Biosystems (Gibson et al., 1996; Heid et al., 1996). Since then over 40 additional Real-Time PCR instruments have been developed by more than a dozen vendors. Because there are so many Real-Time PCR instrument available utilizing a wide range of technologies, scientists face a daunting selection task. The space includes everything from entry level (single color detection, a small number of samples, low cost) to more complex (over 5 channel colors and multiplex detection, thousands of samples processed in each run, and expensive system price). In this chapter we highlight some key features that differentiate Real-Time PCR instruments, with the goal of simplifying the criteria needed to select the instrument that best fit a specific scientist's research needs.
Further reading: PCR Troubleshooting and Optimization: The Essential Guide
from Jan Hellemans and Jo Vandesompele writing in PCR Troubleshooting and Optimization: The Essential Guide:
Real-time quantitative PCR (qPCR) is the gold standard for fast, accurate, sensitive and cost-efficient gene expression analysis. Despite its conceptual simplicity and ease of use, the multi-step qPCR workflow contains many potential pitfalls. An intelligent experiment design and setup, high quality reagents and assays, quality controls in each step of the workflow, proper quantification models and appropriate bio-statistical analyses pave the way to successful gene expression results. This chapter will cover all data analysis aspects from the evaluation of pilot studies and quality controls, through universally applicable quantification models and bio-statistics, to the reporting of experiment results.
Further reading: PCR Troubleshooting and Optimization: The Essential Guide
from Gavin Meredith, Miro Dudas, Mark Landers, Vasiliki Anest, Jonathan Wang, Caifu Chen, Peter Jozsi and Christopher Adams writing in PCR Troubleshooting and Optimization: The Essential Guide:
The field of epigenetics transcends traditional genetics, genomics, molecular biology, and is poised to revolutionize the field of medical research and healthcare. It is a diverse field that encompasses the study of nuclear components such as chromatin structure, including histone modifications, protein/DNA interactions, protein/RNA interactions, and how these factors influence gene function. It also includes the study of DNA methylation and the role that non-coding RNAs play in influencing DNA methylation patterns, chromatin structure and ultimately regulating gene expression. Just as the field of epigenetics is broad and complex, so is the molecular technology of polymerase chain reaction (PCR). For every question one would like to address in any of these areas of epigenetics, there is a PCR application and instrumentation suitable to address it. For example there are numerous PCR-based approaches to look at DNA methylation patterns, densities, and even the methylation status of individual cytosine residues by PCR. Additionally, there are PCR methods to survey ncRNA expression and identify regions of the genome where proteins and RNA interact or where certain functional histone marks are located. This chapter provides an overview of these methodologies with a focus on the advantages and disadvantages of each approach.
Further reading: PCR Troubleshooting and Optimization: The Essential Guide
from Cameron N. Gundry and Matthew D. Poulson writing in PCR Troubleshooting and Optimization: The Essential Guide:
PCR is a highly sensitive and specific technique used in molecular biology laboratories everywhere. It is able to provide near 100% sensitivity and specificity with appropriately designed assays in controlled situations. However, results do not always match this potential. The most common problems in PCR arise from overlooking basic principles in assay design and optimization. Maximum PCR performance depends on key factors which include: 1) choosing an appropriate detection system, 2) using available software for the best primer and probe design, 3) assessing sample quality and controlling inhibitors, 4) avoiding amplicon and environmental contamination, 5) optimizing for reagent quality and concentration, and 6) modifying the thermal cycling protocol for optimal sensitivity and specificity. This chapter will address all of these factors to aid the investigator in designing high quality PCR assays.
Further reading: PCR Troubleshooting and Optimization: The Essential Guide
from N. Reginald Beer and John H. Leamon writing in PCR Troubleshooting and Optimization: The Essential Guide:
PCR has traditionally been performed in microliter-scale reactions because larger scale volumes are prohibitively expensive and wasteful while the smaller scales (nanoliter and below) are impractical with available sample handling tools and detection systems. At the microliter scale, samples can contain mutually competitive and distinct targets, introducing amplification bias and competitive inhibition that degrade assay performance. Microfluidic Emulsion PCR has emerged as a technique to resolve these challenges by a combination of two enabling technologies. Emulsion PCR provides the advantages of fluid partitioning, namely elimination of sample bias and the ability to run millions of reactions in discrete volumes, while microfluidics simultaneously reduces the sample volume, introduces a level of control over emulsion parameters, and provides optical observability of the partitioned microreactors. Furthermore, since microfluidic emulsions can be made monodisperse in size, they allow the assumption of an average dilution per reactor to permit the exploitation of Poisson statistics for very accurate titer estimation. Microfluidic emulsions can also be employed to perform solid-phase amplification with bead-based assays, combining yet another useful technique with the sample partitioning benefits of droplets. We expect the advantages of both emulsion PCR and microfluidics will encourage new applications and the integration of these enabling technologies will improve PCR performance.
Further reading: PCR Troubleshooting and Optimization: The Essential Guide
from Carl T. Wittwer and Jared S. Farrar writing in PCR Troubleshooting and Optimization: The Essential Guide:
The polymerase chain reaction (PCR) has become a fundamental tool in molecular research and clinical testing. The origins of PCR and its early evolution are described, including adaptation to RNA, thermostable polymerases, automation, improvements in specificity and rapid temperature cycling. Perhaps the most significant advance is real-time PCR, combining both amplification and detection into one instrument as a superior solution for nucleic acid quantification. Real-time PCR is enabled by monitoring the reaction with double stranded DNA dyes or specific probes, including hydrolysis, hybridization, and conformation-sensitive probes. Early real-time instruments are compared. PCR product and probe melting analysis continues to improve in resolution, allowing greater sequence detail for genotyping and variant scanning. Microfluidic platforms and digital PCR are destined to find more applications in the future.
Further reading: PCR Troubleshooting and Optimization: The Essential Guide
from John F. Mackay and Carl T. Wittwer writing in PCR Troubleshooting and Optimization: The Essential Guide:
Real-time qPCR using SYBR Green and melting curve analysis to verify specific product amplification has become a standard laboratory technique for rapid, high throughput gene quantification. An extension of this melting curve method - High Resolution melting analysis (HRMA)_ is now doing the same for the analysis of sequence variation, allowing rapid cost-effective discrimination of sequences to SNP level in an automated closed-tube method. Two PCR primers are typically required as with SYBR Green quantification but HRMA differs in its requirement for the use of a saturating dye, precise reaction temperature control and software algorithms to cluster the melting curves. Originally described for SNP analysis (and still the leading application), HRMA is now being used in a wider context- HLA comparisons, microsatellite genotyping and methylation status of DNA sequences. New developments such as unlabeled probes and snapback elements on the PCR primers allow the simultaneous genotyping of a desired SNP with the scanning of the whole amplicon for other sequence variation. This chapter covers some of these developments and provides a guide to those wishing to establish this technique, as well as troubleshooting advice for those already underway.
Further reading: PCR Troubleshooting and Optimization: The Essential Guide
from Jack M. Gallup writing in PCR Troubleshooting and Optimization: The Essential Guide:
One of the least-acknowledged problems with PCR, RT-PCR and qPCR is reaction inhibition. Addressing or eliminating inhibition is central to allowing qPCR to be modeled by the least complex mathematics, and enables more effective troubleshooting of amplifications from difficult templates such as AT- or GC-rich sequences, repetitive sequences, and templates with prohibitive secondary structures. In the absence of inhibition, additives aimed at improving PCR, RT-PCR and qPCR performance can be assessed more directly, allowing investigators to identify and utilize better primer/probe designs, enzymes and master mixes, and formulate better reverse transcription reactions. In addition to inhibition, RNA integrity is another major concern which must be addressed both by using appropriate optical assessments and the 3':5' assay. To address inhibition, commercial kits for removing inhibitory substances have been developed in addition to the SPUD assay and the P-Q assay-development/project-management software tool. Although reagent choice alone plays a large part in determining the success or failure of reverse transcription, PCR, RT-PCR or qPCR, this chapter briefly explores some of the current strategies for detecting, avoiding and/or eliminating inhibition during reverse transcription, PCR, RT-PCR and qPCR. It also discusses strategies to amplify difficult templates and optimize reverse transcription reactions.
Further reading: PCR Troubleshooting and Optimization: The Essential Guide