The ongoing Evolution of qPCR

Methods Vol 50, Issue 4
Pages 215-336  & 
April 2010

edited by  Michael W. Pfaffl

Table of content
Full papers and reviews
Sponsored Application Notes

Transcriptional Biomarkers

Methods Vol 59, Issue 1
Pages 1 - 163  & 
January 2013

edited by  Michael W. Pfaffl

Table of content
Full papers and reviews
Sponsored Application Notes

Advanced Molecular Diagnostics for Biomarker Discovery

Biomolecular Detection and Quantification
Special Issue:
   Advanced Molecular Diagnostics for Biomarker Discovery
   Edited by Michael W. Pfaffl

Volume 5, Pages 1-38 (September 2015) -- Part 1
Volume 6, Pages 1-32 (January 2016) -- Part 2
Download full papers and reviews

Liquid Biopsy & Next Generation Biomarkers

Biomolecular Detection and Quantification
Special Issue:
   Liquid Biopsy & Next Generation Biomarkers
   Edited by Michael W. Pfaffl

Volume 17 (March 2019)
Download full papers and reviews

How to apply the MIQE guidelines - a visual, interactive and practical qPCR & dPCR guide
5th Edition (20th February 2022)
Editors:  Afif M. Abdel Nour & Michael W. Pfaffl
ISBN 9783000488061

Free download via Apple Books (iBooks version) --
Free download via GQ (ePub version) -- ePub (158 MB)

Free download via GQ (PDF version) -- PDF (52 MB)

Please let us know, if you want to participate with an own chapter in the next 6th edition of the MIQE qPCR & dPCR iBook, contact us via

New book chapters in 5th eds:

Chapter 14
Development of a high-throughput data analysis method for evaluating quantitative real-time PCR (qPCR) assays

by Gregory C. Patton, Ph.D., Andrew N. Gray, Ph.D., Nathan A. Tanner, Ph.D., Janine G. Borgaro, Ph.D., Yan Xu, Ph.D., Julie F. Menin, M.S. & Nicole M. Nichols, Ph.D., New England Biolabs, Inc.

Quantitative real-time PCR (qPCR), first demonstrated by Higuchi and colleagues (Higuchi et al., 1992), is an essential molecular biology technique for detecting and quantifying nucleic acids and a mainstay of molecular diagnostics workflows. Workflow simplicity and advances in instrumentation now permit sizeable quantities of data to be generated rapidly, with 96, 384, or even 1536 reactions in a single qPCR experiment. These experiments require thoughtful design and analysis to capture all relevant information, such that accurate and appropriate conclusions can be drawn.
We sought to develop a suite of general use qPCR reagents that maintained performance across various targets and workflow parameters to simplify downstream assay design complexity. This development project involved repeated data collection on a series of test panels, each containing multiple targets. Target were chosen to represent parameters that have been shown to impact assay performance, including variations in primers (e.g., length, GC content, location) and targets (e.g., length, GC content, transcript abundance). Consistent with MIQE guidelines, standard curves were used to evaluate test panel assay performance, but after reviewing efficiency, reproducibility and non-template controls from numerous experiments, it became clear that a more scalable approach to data analysis and visualization was required to better understand how changes in reagent composition impacted performance. To compare various amplicon panels over multiple qPCR runs, instruments, reagents and conditions, a high-throughput data analysis method termed “dots in boxes” was developed. The output of this analysis captures key assay characteristics, including those highlighted in MIQE guidelines, as a single data point for each qPCR target. This method of analysis permits multiple targets and conditions to be compared in a single graph, allowing concise visualization and rapid evaluation of overall experimental success.

Chapter 20
Digital Microfluidic PCR in QIAcuity Nanoplates

Kevin Matthaei, Christina Goebel, Michael Bussmann, Gerald Schock, Afif Abdel Nour, Andreas Missel; QIAGEN GmbH

QIAcuity systems are designed to determine absolute amounts of target DNA in a sample using a digital PCR (dPCR) approach.
Digital PCR uses the procedure of end-point PCR but splits the PCR reaction into many single partitions in which the template is randomly distributed across all available partitions. After PCR, the target molecule is detected by measuring the fluorescence – of either sequence-specific DNA probes or intercalating dyes – in all positive partitions. As the template is distributed randomly, Poisson statistics can be used to calculate the amount of target DNA per positive partition. The total amount of target DNA in all partitions of a well is then calculated by multiplying the amount of target DNA per partition with the number of positive partitions. Measurement of target concentration is determined based on the volume in all analysable partitions, i.e., partitions filled with reaction mix, as identified by a fluorescent dye present in the reaction mix itself. Absolute quantification by dPCR eliminates the need for standard curves to determine amounts of target DNA in a given sample.
Aside from absolute quantification, the QIAcuity Software Suite provides analysis modules for mutation detection, genome editing analysis, copy number variation (CNV), and gene expression analysis.

MIQE & qPCR iBook
How to apply the MIQE guidelines - a visual, interactive and practical qPCR guide
4th Edition (8th June 2020)
Editors:  Afif M. Abdel Nour & Michael W. Pfaffl
ISBN 9783000488061

Free download via iTunes or Apple Books --
Download the interactive PDF -- PDF (47 MB)

Please let us know, if you want to participate with an own application or chapter in the MIQE & qPCR iBook, contact us via


Editorial for 4th edition June 2020

The success story of the qPCR and dPCR MIQE guidelines is continuing!
Editorial for 4th edition published in June 2020
by Afif M. Abdel Nour & Michael W. Pfaffl

Yes the story is continuing! More and more researchers, biological journals, academic and commercial institutions worldwide, are supporting the MIQE guidelines. The very high recitation record of Scopus and Google Scholar documents this. As for June 2020, we count multiple thousand citations for the qPCR MIQE guideline (7.350 by Scopus and 9.800 by Google Scholar) as well as for the digital MIQE guideline (370 by Scopus and 460 by Google Scholar). Hence, the qPCR and dPCR MIQE guidelines are a worldwide standardisation success story, which are driven forward by scientific validity and credibility in the PCR community.
The present 4th edition of the MIQE & qPCR iBook will push the ‘MIQE idea’ even further in any laboratory worldwide and beyond in the scientists’ workflow and minds. It should clearly show how to apply the guidelines and serve as a handy, visual, interactive and practical guide. Since the 2nd edition in July 2016, we provided an additional MIQE & qPCR eBook version, which is readable on any eBook reader device (ePub file). This leaded to more than 4.800 extra downloads of the MIQE & qPCR iBook and eBook from over 40 countries worldwide.
Our goal for the 4th edition is to update the existing content by new hot topics, and to create an overall fancy interactive tool, by interfacing scientific publications with educating pictures, videos and scientific talks. The editors implemented the following new chapters:
  • “ddPCR – droplet digital PCR” authored by Afif Abdel Nour
  • "Reproducible and Sensitive Assays using 3- and 6-colour Crystal Digital PCR™ for Detecting Point Mutations in Human Breast Cancer" authored by scientists from Stilla Technologie
  • "The influence of plastic consumables on qPCR" authored by application specialists by Eppendorf
In summary, we are proud to present a selection of international highly recognized authors from the academic field and from industrial research, presenting their latest research applications. The described qPCR and dPCR methods and applications are tightly linked to the MIQE context, and show it clearly based on educational questionnaires or interactive ‘how to do’ instruction sheets. The at-hand MIQE & qPCR iBook & eBook should deliver the MIQE guidelines directly to the researcher and help to solve the daily problems in the molecular biology laboratory using quantitative PCR, digital PCR, single-cell qPCR, microRNA applications or any comparable techniques using PCR.
The editors hope you like our explanatory, interactive and educational iBook, eBook and ePub concept, showing the advantages of the MIQE guidelines in an easy and understandable way. The proper application and recommendation mentioned in that publication should guarantee the successful qPCR or dPCR application in the lab, and will help authors, reviewers, editors, and researchers to evaluate the quality of the presented publication.

The editors
Afif M. Abdel Nour & Michael W. Pfaffl

MIQE qPCR iBook 3rd edition cover
MIQE & qPCR iBook
How to apply the MIQE guidelines - a visual, interactive and practical qPCR guide
3rd Edition (12th July 2019)
Editors:  Afif M. Abdel Nour & Michael W. Pfaffl
ISBN 9783000488061
Free download via iTunes or Apple Books --
An ePub version will follow asap, early in 2020 :-)
2nd Editions is still available published October 2017 --  Download ePub Version for any eBook reader
Please let us know, if you want to participate with an own application or chapter in the MIQE & qPCR iBook, contact us via

How to apply the MIQE guidelines - a visual, interactive and practical qPCR guide

2nd edition published 26th July 2016
Editors:  Afif M. Abdel Nour & Michael W. Pfaffl
ISBN 9783000488061


MIQE & qPCR Preface:   The qPCR and dPCR MIQE guidelines – A success story!
(Editorial for 2nd edition published in July 2016)

The MIQE guidelines and the resulting scientific validity will be supported by more and more researchers, biological journals, academic and commercial institutions. Today in July 2016 we count more than 4600 citations for the MIQE guideline applied in qPCR and around 160 citations for the digital PCR (dPCR) MIQE guideline (measured by Google Scholar). Hence the qPCR and dPCR MIQE guidelines are a worldwide full success story which will be driven forward by the scientific community, e.g.

The present second edition of the MIQE & qPCR iBook should help to spread this MIQE idea even further in any laboratory worldwide and beyond in the scientists’ workflow and minds. It should clearly show how to apply the guidelines and serve as a handy, visual, interactive and practical guide. For now in the first year after publication of the first edition we could count more than 1200 downloads of the MIQE & qPCR iBook from more than 30 countries. Our goal for the second edition is to update the existing content by new chapters, and to improve this fancy interactive tool, interfacing scientific publications with educating pictures, videos and scientific talks. We implemented multiple new chapters, describing the significance of the reverse transcription reaction, why PCR assay validation is so important for high sensitivity and good reproducibility, and one reviewing chapter about the necessity of performing quality control at all levels in the qPCR workflow. In summary we are proud to present a selection of international highly recognized authors from the academic field as well from industrial research presenting their latest applications. Described qPCR / dPCR methods and applications are linked to the MIQE context and show it on the basis of educational questionnaires or interactive ‘how to do’ instruction sheets. The at-hand MIQE & qPCR iBook should deliver the MIQE guidelines directly to the researcher and help to solve the daily problems in the molecular biology laboratory using quantitative PCR, digital PCR, single-cell qPCR, microRNA applications or any comparable techniques using PCR.

We hope you like our explanatory, interactive and educational iBook concept, showing the advantages of the MIQE guidelines in an easy and understandable way, and to guarantee the successful qPCR or dPCR application at the lab bench.

The editors
Afif M. Abdel Nour & Michael W. Pfaffl

New Chapters in 2nd edition:
  • Editorial
  • Reverse Transcription
  • qPCR Assay validation
  • Quality control in qPCR

MIQE & qPCR iBook
MIQE & qPCR iBook
How to apply the MIQE guidelines - a visual, interactive and practical qPCR guide

1st edition published 11th May 2015
Editors:  Afif M. Abdel Nour & Michael W. Pfaffl
ISBN 9783000488061

Throughout the past 30 years Polymerase Chain Reaction (PCR) has proven to be the most powerful technique in a scientist toolbox. Only few techniques had a comparable success story like PCR. This iBook will guide you in better practicing in your laboratory thanks to the MIQE guideline.

MIQE & qPCR iBook – a digital publication: How making the MIQE guidelines easier to follow.
Afif M. Abdel Nour  &  Michael W. Pfaffl
Presentation at qPCR & NGS 2015 Symposium --

Chapters in 1st edition:
  • Preface
  • Introduction & The history of MIQE
  • Quantification strategies
  • RNA quality control
  • qPCR oligonucleotides / Primer design software
  • qPCR instrumentation
  • Selection of reference genes      
  • Data analysis & statistics
  • MIQE compliant qPCR workflow control
  • MIQE guidelines and microRNA
  • Digital PCR Applications and Assays
  • MIQE compliance using qPCR arrays
  • MIQE translations & online applications
  • Credits & Imprint
  • Afif Abdel Nour & Michael W. Pfaffl
  • Stephen Bustin et al.
  • MW Pfaffl
  • Eva Schmidt et al.
  • James Flynn et al.
  • Nick Newton et al.
  • Philip Zimmermann et al.
  • Mikael Kubista, Amin Forootan et al.
  • M Hren, K Zupancic, T Berto et al.
  • Ditte Andreasen, et al.
  • Yann Jouvenot & Viresh Patel
  • Greg Shipley
  • Afif Abdel Nour
  • Afif Abdel Nour & Michael W. Pfaffl

Please let us know, if you want to participate with an own application or chapter in the MIQE & qPCR iBook.
Contact us via

Welcome to Stephen Bustin's publishing website

an iTunes eBook series  Definitive qPCR  edited by Stephen Bustin

About the qPCR Expert Series
This constitutes a series of books that are up-to-date and easily affordable for students and filled with honest opinions about qPCR initially, but expanding to deal with other molecular techniques. Each technique is placed into its historical context and the books explain why certain procedures are carried out the way they are, and identify the critical issues that determine the success or failure of an experimental procedure. Electronic media are ideal for this purpose, as update can be issued within minutes of a new instrument, protocol, enzyme or method becoming available. From the reader’s point of view, the fact that this update is going to be free means that he/she will always have the latest information at his/her finger tips with the added security that the information has been vetted and verified prior to being included in the e-book.

Conventional publishing has two big disadvantages:
  • the time from completing the writing of a book to it being published can be a long one. In an area as fast-moving as molecular biology, this means that concepts, protocols and instruments may be well past their sell-by date by the time a book appears.
  • academic publications are very expensive and certainly beyond the financial reach of many students and post-doctoral researchers, particularly in developing countries. Whatever the reason for the high prices, they mean that the individuals most in need of advice find it difficult to obtain. Furthermore, many peer-reviewed journals focussing on techniques are not freely accessible, posing further problems for advice seekers.
The Expert Series breaks down each technique into its constituent parts and provides exhaustive information about that sub section. So if someone is interested in qPCR in general, without feeling the need to explore this technology in depth, they can buy the Basic Principles, which will give them all the information they require but not get bogged down in detail about which analysis method is the best the use under what circumstances. Conversely, someone already au fait with the topic might be more interested in Assay Optimisation and obtain all the relevant information from that volume.

Each book will be issued in two formats: iBooks that can be purchased through Apple's iTunes store in around 50 countries as well as in a PDF format, which can be bought in any country around the world using a secure site and credit card payment.

Definitive qPCR: A practical Guide

This volume deals with the practical steps required to characterise, optimise and validate a qPCR assay. It deals with all aspects of laboratory organisation, instruments, pipettes, plasticware and reagents as well as providing protocols for optimisation of annealing temperatures, primer concentration and determining assay efficiency. The 320 pages are richly illustrated with examples, tips, explanations and all information is hyperlinked to provide access to the huge amount of information that is available online, but not always found in obvious places.
This book will be invaluable to anyone wanting to design, validate or trouble shoot qPCR assays. Its easily accessible language takes the reader through the complex steps that make up a successful qPCR assay and will result, it is hoped, in data that are accurate, robust and biologically relevant.

Definitive qPCR: Basic Principles
ISBN 978-0-9572024-1-2
This book provides a detailed insight into the principles of the real-time quantitative polymerase chain reaction (qPCR). The narrative places the protocols, reagents and instruments into their historical and scientific contexts and provides a detailed explanation of the concepts that have made this technology the enabling molecular technology par excellence. The different chapters describe the background to oligonucleotide synthesis, DNA polymerases, trace the route from endpoint to real-time PCR and explain the basics of fluorescence and instrumentation. The book then describes the various chemistries used in qPCR in individual chapters dealing with DNA-binding reporters, primer/fluorochrome combination as well as structured and unstructured probes. The final chapter deals with MIQE and describes the events that led to its realisation. An appendix lists reagents, provides a summary of all qPCR instruments on the market to-day and lists a wide range of web-based resources. The book is illustrated throughout with detailed diagrammes and is targeted both at aspiring as well as experienced users of this ubiquitous technology.

1.    PCR
1.1.    Introduction
1.2.    Oligonucleotide synthesis
1.3.    Reagents
2.    Real-time PCR
2.1.    The road to qPCR
2.2.    Fluorescence
2.3.    Instrumentation
3.    Chemistries
3.1.    DNA-binding reporters
3.2.    Primer/fluorophore combinations
3.3.    Unstructured probes
3.4.    Allosteric toggle probes
4.    MIQE
4.1.    Measles virus and gut pathology
4.2.    The case for MIQE
5.    Appendices

Definitive qPCR: Assay Design
ISBN 978-0-9572024-0-5
This book provides an exhaustive guide to assay design for quantitative real-time PCR. The book describes the basic concepts important for amplicon selection and primer and probe design. There are step-by-step examples for designing probe-based and SYBR Green assays targeting mRNA and fungal pathogens using several popular design programs. These are then exposed to extensive in silico analysis to identify the optimum amplicon/primer/probe combination. There is a detailed trouble-shooting guide, a listing of instruments, reagents and additional information available on the internet, all with hyperlinks. In addition, Keynote presentations (iBook format only) summarise the main concepts of standard assay design, multiplex assay design and explaining the rationale behind MIQE, the guidelines for qPCR publication transparency.

1.    Introduction
1.1.    Why assay design is important
1.2.    Why design your own assay?
1.3.    Tools for assay design
2.    Amplicons
2.1.    Amplicon selection – mRNA
2.2.    Amplicon selection – pathogen
3.    Primers and Probes
3.1.    Design – mRNA
3.2.    In silico validation – mRNA
3.3.    Design and in silico validation-pathogen
3.4.    Multiplex PCR
4.    What can go wrong?
5.    Appendices
5.1.    Design principles and comparisons
5.2.    Reagents & Instrumentation
5.3.    MIQE
5.4.    Web-based resources

Definitive qPCR: Nucleic Acid Quality Control
ISBN 978-0-9572024-4-3
The assessment of nucleic acid purity and integrity is one of the essential steps preceding microarray, qPCR and sequencing experiments. Both quality parameters have direct effects on the reliability, accuracy and biological/clinical relevance of results obtained using these molecular techniques.
This book discusses both provides a detailed overview of the important evaluation criteria that make up successful nucleic acid quality control, with the main focus on RNA. It traces the historical context of nucleic acid extraction, describes prokaryotic and eukaryotic in vivo RNA degradation mechanisms, summarises information about the different instruments used to assess nucleic acid quality and analyses RNA and DNA purity as well as RNA integrity in detail. The appendices contain a general illustration of some of the molecular methods that rely on high quality nucleic acids and a set of figures making the case for MIQE, the guidelines laid out for qPCR assay design and publication.
The book is extensively referenced and hyperlinked to additional sources of information. It is ideal for anyone wanting to find all relevant information about nucleic acid quality assessment in one convenient place.

1.    The Basics
1.1.    Introduction
1.2.    Nucleic acid extraction
1.3.    RNA and a little DNA
2.    Instruments
2.1.    Nano Spectrophotometers
2.2.    Lab on a chip
2.3.    Agilent Bioanalyser 2100
2.4.    BioRad Experion
2.5.    Agilent 2200 tapestation et al
3.    Quality
3.1.    Introduction
3.2.    Inhibition
3.3.    Integrity
4.    Appendices
4.1.    Protocols
4.2.    In vitro measurements
4.3.    MIQE
4.4.    Reagents and Instrumentation
4.5.    Web-based information

New releases - Available over the next few months (~January, May and August 2013)


Horizon Scientific Press | Caister Academic Press | PCR Books

Our high level PCR books bring together expert international authors under the skilled editorship of leading scientists to produce state-of-the-art compendiums of current research. Aimed at the research scientist, graduate student, medical reseacher and other professionals, these books are highly recommended for all PCR laboratories. Every PCR, microbiology and bioscience library should have a copy of each of the following books.

Edited by: Jim Huggett and Justin O'Grady
Published: 2014
Hardback: ISBN 978-1-908230-41-6 £159, $319. Ebook: ISBN 978-1-908230-64-5 £159, $319
The editors of this book have commissioned an excellent series of chapters representing two key molecular diagnostic areas: cancer and infectious diseases. The cancer section deals with the challenges in identifying genetic, epigenetic and transcriptomic biomarkers. The infectious disease section describes the current clinical applications of molecular diagnostics for the detection of viral, bacterial and fungal pathogens as well as an example of the use of molecular diagnostics outside the clinic environment. A cautionary tale describing what can go wrong when molecular methods are applied incorrectly is also provided and makes fascinating reading. A substantial component of the book is dedicated to the process of translating a preclinical test to the bedside and describes the progress in the near patient point-of-care molecular diagnostics market. This is a fundamental consideration for successful translation of diagnostics tests from bench to bedside and is crucial for molecular diagnostics to have an impact on patient care. The final chapter offers a prediction of future trends in the molecular diagnostics of infectious diseases. This volume is essential reading for anyone involved in the development or application of molecular diagnostics and is recommended for all clinical diagnostics laboratories. read more ...

Edited by: Nick A. Saunders and Martin A. Lee
Published: 2013
Hardback: ISBN 978-1-908230-22-5 £159, $319. Ebook: ISBN 978-1-908230-87-4 £159, $319
This essential manual provides both the novice and experienced user with an invaluable reference to a wide-range of real-time PCR technologies and applications and provides an overview of the theory of this increasingly important technique. Renowned international authors present detailed technical insights into the underlying principles, methods and practice of real-time PCR. The initial chapters cover the important aspects of real-time PCR including choosing an instrument and probe system, set-up, nucleic acid synthesis, sample extraction controls, and validation and data analysis. Further chapters provide a comprehensive overview of important real-time PCR methodologies such as quantification, expression analysis and mutation detection. This is complemented by the final chapters, which address the application of real-time PCR to diagnosis of infectious diseases, biodefence, veterinary science, food authenticity and molecular haplotyping. This timely and authoritative volume serves both as a basic introduction to real-time PCR and as a source of current trends and applications for those already familiar with the technology. The editors also aim to stimulate readers of all levels to develop their own innovative approaches to real-time PCR. An essential book for all laboratories using PCR. read more ...
Real-Time PCR in Food
                                      Science: Current Technology and

Edited by: David Rodriguez-Lazaro
Published: 2013   ISBN: 978-1-908230-15-7
Price: GB £159 or US $319
Written by experts in the field, this book is an indispensable manual for scientists in the food industry. The first section provides an introduction to real-time PCR, discusses the use of PCR diagnostics in food science, describes the principles and methods of sample preparation, and covers the verification and control of PCR procedures. The eleven chapters in the second section cover the use of real-time PCR to detect various pathogens including Salmonella, Listeria, E. coli, Campylobacter, Yersinia, Staphylococcus, Clostridium, viruses and parasites. Also included is a chapter on the standardisation of real-time PCR methods in food microbiology. In the final section authors cover the use of real-time PCR for the analysis of genetically modified organisms, food allergens and for identification of animal or plant species. An invaluable book for anyone involved in food microbiology or the detection of foodborne pathogens and a recommended volume for all microbiology laboratories. read more ...
Quantitative Real-time PCR in
                                      Applied Microbiology

Edited by: Martin Filion
Published: 2012   ISBN: 978-1-908230-01-0
Price: GB £159 or US $319
Written by experts in the field and aimed specifically at microbiologists, this volume describes and explains the most important aspects of current qPCR strategies, instrumentation and software. Renowned authors cover the application of qPCR technology in various areas of applied microbiology and comment on future trends. Topics covered include instrumentation, fluorescent chemistries, quantification strategies, data analysis software, environmental microbiology, water microbiology, food microbiology, gene expression studies, validation of microbial microarray data and future trends in qPCR technology. The editor and authors have produced an outstanding book that will be invaluable for all microbiologists. A recommended book for all microbiology laboratories. read more ...
PCR Troubleshooting and
                                      Optimization: The Essential Guide

Edited by: Suzanne Kennedy and Nick Oswald
Published: 2011   ISBN: 978-1-904455-72-1
Price: GB £159 or US $319

This book discusses the strategies for preparing effective controls and standards for PCR, when they should be employed and how to interpret the information they provide. It highlights the significance of optimization for efficiency, precision and sensitivity of PCR methodology and provides essential guidance on how to troubleshoot inefficient reactions. Experts in PCR describe design and optimization techniques, discuss the use of appropriate controls, explain the significance of standard curves and explore the principles and strategies required for effective troubleshooting. Authors highlight the importance of sample preparation and quality, primer design, controlling inhibitors, avoiding amplicon and environmental contamination, optimizing reagent quality and concentration, and modifying the thermal cycling protocol for optimal sensitivity and specificity. In addition, specific chapters discuss the history of PCR, the choice of instrumentation, the applications of PCR in metagenomics, high resolution melting analysis, the MIQE guidelines, and PCR at the microliter scale. The strategies, tips and advice contained in this concise volume enable the scientist to optimize and effectively troubleshoot a wide range of techniques including PCR, reverse transcriptase PCR, real-time PCR and quantitative PCR. An essential book for anyone using PCR technology. read more ...
PCR Troubleshooting and
                                      Optimization: The Essential Guide

Chapter 5 - RT-PCR Optimisation Strategies
by Martina Reiter & Michael W. Pfaffl
Physiology Weihenstephan, Weihenstephaner Berg 3, 85354 Freising, Germany

PCR technology is based on a simple principle; an enzymatic reaction that increases the initial amount of nucleic acids. This method makes it possible to detect specific mRNA transcripts in any biological sample. Performing RT-PCR analysis does not only comprehend this experimental PCR step. Following the whole workflow of a RT-PCR quantitative analysis, it starts with the sampling step, followed by nucleic acid extraction and stabilization, cDNA synthesis and finally the qPCR where the mRNA quantification takes place. Problems arise when optimization of the experimental work flow becomes necessary because of high technical variations. The PCR reaction itself is a quite stable reaction with reproducibility between 2-8%. Therefore the source of experimental variances can often be found in the pre-PCR analytical steps. Usually this is neglected and optimization is done for PCR reaction only. In this chapter – RT-PCR optimization strategies - the whole workflow of RT-PCR experiment will be discussed, because the identification of the source of variability is only possible following error accumulation in every single step. Reliable data can be created when the technical variance caused by the experimental steps is kept as low as possible. In this chapter many recommendations to decrease the technical variance can be found.
Lab-on-a-Chip Technology:
                                      Biomolecular Separation and

Edited by: Keith E. Herold and Avraham Rasooly
Published: 2009   ISBN: 978-1-904455-47-9
Price: GB £159 or US $319
The editors of this book have brought together expert authors from many countries to produce a comprehensive volume focusing on the applications of LOC technology in the biomedical and life sciences. The first section includes chapters on LOC biomolecule separation. Separation of biomolecules is an important element of various clinical laboratories and is required for many "down stream" analytical applications. Various electrophoresis and liquid chromatography applications for proteins and DNA are described as well as methods for cell separation, with an emphasis on blood cell separation, which have many important clinical applications. The second part includes chapters on analysis and manipulation technologies. Authors describe protein, genetic (mainly PCR) and transcriptome analysis with examples from research and clinical applications, as well as cell manipulation and analysis including cell viability analysis and microorganism capturing. A skillful selection of topics of exceptional importance to current science ensures that this book will be of major value to a wide range of molecular biologists, clinical scientists, microbiologists, biochemists and anyone interested in LOC technology or developing applications for LOC devices. read more ...
Lab-on-a-Chip Technology:
                                      Fabrication and Microfluidics

Edited by: Keith E. Herold and Avraham Rasooly 
Published: 2009   ISBN: 978-1-904455-46-2
Price: GB £159 or US $319 
This invaluable book describes the latest methods and novel technologies being developed for the fabrication of LOC devices and new approaches for fluid control and manipulation. Expert authors from around the world describe and discuss the newest technologies for the prototyping of devices, including replication and direct machining methods of fabrication. Part I of the book covers all aspects of fabrication including laser micromachining, silicon and glass micromachining, PMMA and COC microfluidic substrates, and xurography (LOC prototyping with a cutting plotter). Part II focuses on fluid control and manipulation for LOC systems. As well as providing examples of the use of pumps in microfluidics, topics covered include electrokinetic pumping (electroosmois), electrochemical pumping and electrowetting, and the fabrication of a microchip for rapid polymerase chain reaction (PCR). This comprehensive volume presents the current technologies in the field and includes theoretical and technical information to enable both the understanding of the technology and the reproduction of experiments. The book aims to help the reader to understand current LOC technologies, to perform similar experiments, to design new LOC systems and to develop new methodologies and applications. An essential book for biologists and clinicians using LOC technology and developing applications and also for engineering, chemical and physical science researchers developing analytical technologies. The book will also be useful as a teaching tool for bioengineering, biomedical engineering and biology. read more ...

Edited by: Julie Logan, Kirstin Edwards and Nick Saunders
Published: 2009   ISBN: 978-1-904455-39-4
Price: GB £159 or US $319 
This essential manual presents a comprehensive guide to the most up-to-date technologies and applications as well as providing an overview of the theory of this increasingly important technique. Renowned experts in the field describe and discuss the latest PCR platforms, fluorescent chemistries, validation software, data analysis, and internal and external controls. This timely and authoritative volume also discusses a wide range of RT-PCR applications including: clinical diagnostics, biodefense, RNA expression studies, validation of array data, mutation detection, food authenticity and legislation, NASBA, molecular halotyping, and much more. An essential book for all laboratories using PCR. read more ...
                                      PCR in Microbiology: From
                                      Diagnosis to Characterization

Edited by: Ian M. Mackay
Published: 2007   ISBN: 978-1-904455-18-9
Price: GB £159 or US $319
The editor and authors have produced an excellent book that will be extremely useful for all microbiologists. A recommended book for all microbiology laboratories. read more ...
                                      Troubleshooting: The Essential

Author: Michael L. Altshuler
Published: 2006   ISBN: 978-1-904455-07-3
Price: GB £30 or US $60
A unique PCR troubleshooting guide that is an essential companion for anyone who uses the polymerase chain reaction technique. Aimed at a reader with some experience in PCR the book discusses the many and varied problems encountered with PCR together with tips, advice and procedures to obviate rather than overcome the PCR problems. Written in the language of the laboratory with a little humour and a down-to-earth approach, the book is easy to read and understand. If you fail at PCR, consult this book! The advice in these pages is invaluable and is the sort of advice that is not usually found elsewhere. In the words of the author, remember that there are many other things in life than PCR ... for example, isothermal DNA amplification. read more ...

Book cover A-Z of qPCR

A-Z of Quantitative RCR

IUL Biotechnology Series

ISBN: 0-9636817-8-8

US$ 119.95
Flyer for the book   =>  PDF

This book is a comprehensive manual to allow both the novice researcher and the expert to set up and carry out quantitative PCR assays from scratch. However, this book also sets out to explain as many features of qPCR as possible, provide alternative viewpoints, methods, and aims to simulate the researchers into generating, interpreting, and publishing data that are reproducible, reliable, and biologically meaningful.

Search inside  @

Real Time PCR
BIOS - Advanced Methods

by Tevfik Dorak

Real-time PCR is based on the conventional principles of PCR and, with the simple shift of emphasis from the end-product to the whole course of the PCR process, has established itself as the most sensitive and specific quantitative PCR method. Real-time PCR is, like any other modern method in molecular genetics, expanding, with potential applications even in proteomics.

With a variety of detection chemistries, an increasing number of platforms, multiple choices for analytical methods and the jargon emerging along with these developments, real-time PCR is facing the risk of becoming an intimidating method, especially for beginners. Real-Time PCR provides the basics, explains how they are exploited to run a real-time PCR assay, how the assays are run and where these assays are informative in real life. It doesn't cover every aspect of real-time PCR, but addresses the most practical aspects of the techniques with the emphasis on 'how to do it in the laboratory'. Keeping with the spirit of the Advanced Methods Series, most chapters provide an experimental protocol as an example of a specific assay.

The BIOS Advanced Methods series is intended for advanced undergraduates, graduate students and established research scientists. Titles in this series are designed to cover current important areas of research in life sciences, and include both theoretical background and detailed protocols. The aim is to give researchers sufficient theory, supported by references, to take the given protocols and adapt them to their particular experimental systems.

Early, rapid and sensitive veterinary molecular diagnostics - real time PCR applications
Erika Pestana, Sandor Belak, Adama Diallo, John R. Crowther, Gerrit J. Viljoen
2010, 310 pages, Englisch  Springer Netherland

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This book gives a comprehensive account of the practical aspects of Real time PCR and its application to veterinary diagnostic laboratories. The optimisation of assays to help diagnose livestock diseases is stressed and exemplified through assembling standard operating procedures from many laboratory sources. Theoretical aspects of PCR are dealt with as well as quality control features necessary to maintain an assured testing system. The book will be helpful to all scientists involved in diagnostic applications of molecular techniques, but is designed primarily to offer developing country scientists a collection of working methods in a single source. The book is an adjunct to the Molecular Diagnostic PCR Handbook published in 2005.

The PCR Revolution  (2010)
Cambridge University Press
ISBN: 978-0521882316
The invention of the polymerase chain reaction (PCR) won the Nobel Prize in Chemistry in 1994 and remains one of the most important scientific discoveries of the twentieth century. More than 50,000 researchers in the United States use PCR replication technology, and yet a book has not been published on the subject in more than ten years. In this book, Dr. Stephen A. Bustin, a world-renowned PCR expert, examines in detail the latest innovations and the overall impact of PCR on many areas of molecular research. The book contains personal reflections, opinions, and comments by leading authorities on the many applications of the PCR and how this technology has revolutionized their respective areas of interest. This book conveys the ways in which PCR has overcome many obstacles in life science and clinical research and also charts the PCR's development from time-consuming, low throughput, non-quantitative procedure to today's rapid, high throughput, quantitative super method.

Contents -

PCR Technology: Current Innovations, Third Edition

Tania Nolan, Sigma Life Science Custom Products, Haverhill, UK
Stephen A. Bustin, Anglia Ruskin University, Cambridge, UK
Published: June 13, 2013 by CRC Press
Content:457 Pages | 161 Illustrations
ISBN 9781439848050

PCR’s simplicity as a molecular technique is, in some ways, responsible for the huge amount of innovation that surrounds it, as researchers continually think of new ways to tweak, adapt, and re-formulate concepts and applications. PCR Technology: Current Innovations, Third Edition is a collection of novel methods, insights, and points of view that provides a critical and timely reference point for anyone wishing to use this technology.

  • Covers all aspects of PCR and real-time PCR
  • Uses an accessible writing style appealing to a broad range of readers
  • Examines buffer and oligonucleotide components
  • Discusses assay design and instruments
  • Presents a plethora of applications and innovations
  • Supplies detailed "how to" information and protocols, along with tips and tricks from recognized experts
Topics in this forward-thinking volume include:
  • The purification and handling of PCR templates
  • The effect of the manufacture and purification of the oligonucleotide on PCR behavior
  • Optimum buffer composition
  • Probe options
  • The design and optimization of qPCR assays
  • Issues surrounding the development and refinement of instrumentation
  • Effective controls to protect against uncertainties due to reaction variability
  • Covering all aspects of PCR and real-time PCR, the book contains detailed protocols that make it suitable as both a reference and an instruction manual. Each chapter presents detailed guidelines as well as helpful hints and tips supplied by authors who are recognized experts in their fields. In addition to descriptions of current technology and best practices, the book also provides information about new developments in the PCR arena.

Quantitative Real-Time PCR: Methods and Protocols   (Methods in Molecular Biology)
by Roberto Biassoni, Alessandro Raso

Quantitative Real-Time PCR: Methods and Protocols focuses on different applications of qPCR ranging from microbiological detections (both viral and bacterial) to pathological applications. Several chapters deal with quality issues which regard the quality of starting material, the knowledge of the minimal information required to both perform an assay and to set the experimental plan, while the others focus on translational medicine applications that are ordered following an approximate logical order of their medical application. The last part of the book gives you an idea of an emerging digital PCR technique that is a unique qPCR approach for measuring nucleic acid, particularly suited for low level detection and to develop non-invasive diagnosis. Written for the Methods in Molecular Biology series, most chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, laboratory protocols and tips on troubleshooting and avoiding known pitfalls.
Practical and authoritative, Quantitative Real-Time PCR: Methods and Protocols aims to aid researchers seeking to devise new qPCR-based approaches related to his or her area of investigation.

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Product Details
Series: Methods in Molecular Biology (Book 1160)
Hardcover: 231 pages
Publisher: Humana Press; 2014 edition (May 31, 2014)
Language: English
ISBN-10: 1493907328

Chapter 5 -- mRNA and microRNA Purity and Integrity:  The Key to Success in Expression Profiling
Benedikt Kirchner, Vijay Paul, Irmgard Riedmaier and Michael W. Pfaffl
Quantitative Real-Time PCR: Methods and Protocols   (Methods in Molecular Biology)
by Roberto Biassoni, Alessandro Raso

RNA quality control is a crucial step in guaranteeing integer nondegraded RNA and receiving meaningful results in gene expression profiling experiments, using micro-array, RT-qPCR (Reverse-Transcription quantitative PCR), or Next-Generation-Sequencing by RNA-Seq or small-RNA Seq. Therefore, assessment of RNA integrity and purity is very essential prior to gene expression analysis of sample RNA to ensure the accuracy of any downstream applications. RNA samples should be nondegraded or fragmented and free of protein, genomic DNA, nucleases, and enzymatic inhibitors. Herein we describe the current state-of-the-art RNA quality assessment by combining UV/Vis spectrophotometry and microfl uidic capillary electrophoresis.

Chapter 15 -- Posttranscriptional Regulatory Networks:  From Expression Profi ling to Integrative Analysis of mRNA and MicroRNA Data
Swanhild U. Meyer, Katharina Stoecker, Steffen Sass, Fabian J. Theis and Michael W. Pfaffl
Quantitative Real-Time PCR: Methods and Protocols   (Methods in Molecular Biology)
by Roberto Biassoni, Alessandro Raso

Protein coding RNAs are posttranscriptionally regulated by microRNAs, a class of small noncoding RNAs. Insights in messenger RNA (mRNA) and microRNA (miRNA) regulatory interactions facilitate the understanding of fi ne-tuning of gene expression and might allow better estimation of protein synthesis. However, in silico predictions of mRNA–microRNA interactions do not take into account the specifi c transcriptomic status of the biological system and are biased by false positives. One possible solution to predict rather reliable mRNA-miRNA relations in the specifi c biological context is to integrate real mRNA and miRNA transcriptomic data as well as in silico target predictions. This chapter addresses the workfl ow and methods one can apply for expression profi ling and the integrative analysis of mRNA and miRNA data, as well as how to analyze and interpret results, and how to build up models of posttranscriptional regulatory networks.

microRNA Books

microRNA: Medical Evidence -- From Molecular Biology to Clinical Practice
Editors: Santulli, Gaetano (Ed.)
eBook -- Advances in Experimental Medicine and Biology, 2015

This volume explores microRNA function in a wide array of human disorders, providing a clinical basis for precision medicine and personalized therapies using these molecules. The twenty-one chapters, all authored by internationally-renowned experts, open with an introduction contextualizing microRNA manipulation within today’s initiatives towards precision medicine. The following chapters explore the clinical role of microRNAs in the diagnosis and treatment of metabolic and cardiovascular disorders, focusing on mitochondrial fitness, arterial hypertension, cardiovascular remodeling, cerebrovascular disease, pulmonary hypertension, diabetic kidney disease, and kidney transplantation. The subsequent chapters discuss the importance of microRNAs in the wound healing process and in skin disease, in the pathogenesis of allergy, in human ovulation, and in infection. The book concludes with chapters  which outline the emerging role of microRNAS in doping and detail microRNA profiling.

MicroRNAs -- From Basic Science to Disease Biology
Edited by Krishnarao Appasani
Cambridge University Press August 2009 ISBN: 9780521118552

MicroRNAs (miRNAs) are RNA molecules, conserved by evolution, that regulate gene expressions and their recent discovery is revolutionising both basic biomedical research and drug discovery. Expression levels of MiRNAs have been found to vary between tissues and with developmental stages and hence evaluation of the global expression of miRNAs potentially provides opportunities to identify regulatory points for many different biological processes. This wide-ranging reference work, written by leading experts from both academia and industry, will be an invaluable resource for all those wishing to use miRNA techniques in their own research, from graduate students, post-docs and researchers in academia to those working in R&D in biotechnology and pharmaceutical companies who need to understand this emerging technology. From the discovery of miRNAs and their functions to their detection and role in disease biology, this volume uniquely integrates the basic science with industry application towards drug validation, diagnostic and therapeutic development. Forewords by: Sidney Altman, Yale University, Winner of the Nobel Prize in Chemistry, 1989 and Victor R. Ambros, Dartmouth Medical School, Co-discoverer of MicroRNAs.

MicroRNAs in Medicine
Editor(s): Charles H. Lawrie
Published Online: 1 Nov 2013,  Online ISBN:  9781118300312

MicroRNAs in Medicine provides an access point into the current literature on microRNA for both scientists and clinicians, with an up-to-date look at what is happening in the emerging field of microRNAs and their relevance to medicine. Each chapter is a comprehensive review, with descriptions of the latest microRNA research written by international leaders in their field. Opening with an introduction to what microRNAs are and how they function, the book goes on to explore the role of microRNAs in normal physiological functions, infectious diseases, non-infectious diseases, cancer, circulating microRNAs as non-invasive biomarkers, and finally their potential as novel therapeutics.
Including background information on the field as well as reviews of the latest research breakthroughs, MicroRNAs in Medicine is a one-stop source of information to satisfy the specialists and non-specialists alike, appealing to students, researchers, and clinicians interested in understanding the potential of microRNAs in medicine and research.

Free Nature reprint collection: MicroRNA from bench to clinic
Read the collection online

Since their discovery, microRNAs have revolutionized cell biology and completely changed the way we view the regulation of gene expression. These short regulating RNAs are now known to be involved in all cellular and developmental pathways and all major types of disease so far investigated. This Nature Reprint Collection presents some of the recent advances in moving microRNAs from basic research into the clinic both as diagnostic biomarkers and therapeutic targets.
Read the collection and learn more about:
  •     microRNA qPCR profiling to identify minimally invasive biomarkers in biofluids
  •     The prognostic importance of microRNA in situ hybridization against miR-21
  •     The potential of microRNAs as therapeutic targets in cardiovascular disease, diabetes and epilepsy

MicroRNA and Cancer -- Methods and Protocols
Humana Press 2011
Edited by Wei Wu; Institute for Biocomplexity and Informatics, Department of Biological Science,
University of Calgary, Calgary, AB, Canada

The discovery of microRNAs (miRNAs or miRs) heralded a new and an exciting era in biology and started a new chapter in human gene regulation. The miRNAs, a class of small endogenous noncoding RNAs (~22 nt), fine tune the gene expression at the posttranscriptional level through mainly binding 3′-UTR of mRNAs. They are involved in stem cell self-renewal, cellular development, differentiation, proliferation, and apoptosis. Small miRNAs have big impacts in cancer development. Among the many miRNAs, a subset of miRNAs were identified as regulators of neoplastic transformation, tumor progression, invasion, and metastasis as well as tumor-initiating cells (cancer stem cells). The widespread deregulation of miRNomes in diverse cancers when compared to normal tissues have been unveiled. The oncomirs (oncogenic miRNAs), TSmiRs (tumor suppressive miRNAs), and MetastamiRs (miRNAs associated to cancer metastasis) comprise an importantpart of the cancer genome and confer pivotal diagnostic and prognostic significance. Moreover, cancer associated miRNAs are proving worthwhile for developing effective cancer biomarkers for individualized medicine and potential therapeutic targets. This book provides the latest and foremost miRNAs knowledge of cancer research applications from scientists all over the world. It is organized in two parts: the first part begins with a general introduction of miRNA biogenesis which is followed by chapters on the computational prediction of new microRNAs in cancer, the innovative technologies for modulating miRNAs of interests, and an overview of miRNA-based therapeutic approaches for cancer treatment. The second part of the book provides experimental and computational procedures in miRNA detection with diverse techniques, miRNA library construction, epigenetic regulation of miNRAs, microRNA::mRNA regulatory networks predicted with computational analysis in cancer cells or tissues, and the principle of designing the miRNA-mimics for miRNA activation. These chapters have been written for practical use in laboratories for graduate students, postdoctoral fellows, and scientists in cancer research. The contributors have shared their most valuable experiences in the translation of miRNA knowledge into cancer research. MicroRNA is a fast growing field, and microRNA knowledge is a pivotal element of cancer biology. An individual miRNA interferes with a broad range of mRNAs; conversely, a single mRNA could be targeted by a variety of miRNAs. The complexity of miRNA::mRNA interaction is far-reaching and a bit beyond our understanding to date. This book is expected to provide the basic principles of experimental and computational methods for microRNA study in cancer research and provide a firm grounding for those who wish to develop further applications. I am especially indebted to Prof. Shu Zheng and Dr. Suzanne D. Conzen for giving me the opportunity to gain substantial experience in cancer research. I would like to thank Dr. Gunglin Li for his kind support. Without their confidence and continuous support, many things would not have been possible. I also thank Prof. John Walker for his encouragement. Finally, I am grateful to all of the contributing authors for providing such high quality manuscripts.

microRNA maturation -- Methods and Protocols
Humana Press 2014
Edited  by  Christoph Arenz
Institute  for  Chemistry,  Humboldt-Universität  zu  Berlin,  Berlin,  Germany

The last decade has seen a dramatic development in the fi eld of micro RNAs (miRNAs). Starting with a small set of small noncoding regulatory RNAs in D. melanogaster and C. elegans miRNAs are now regarded as important regulatory components in many eukaryotic species including humans. The number of reported miRNAs exceeds 1,000 and many of these miRNAs have been implicated in important biological processes and human diseases. In this volume of “Methods in Molecular Biology” we concentrate on the pathway of microRNA maturation. After its synthesis, the primary miRNA transcript (pri-miRNA) is cleaved by indonuclease called Drosha to yield the precursor miRNA (pre-miRNA). After being transported to the cytoplasm, the latter is further cleaved by another nuclease called Dicer to yield the mature double-stranded miRNA. This mature miRNA consists of the active guide strand which remains bound to the miRNA effector complex, whereas the passenger strand is degraded. These few relatively simple steps (although the cellular machinery promoting this pathway is rather complex) are common to most miRNAs and thus are highly important to study. In this book we concentrate on three important aspects of this maturation pathway: First of all, we give an overview over the current knowledge of the pathway of miRNA maturation (Chapter 1 ) and how this pathway relates to human disease (Chapter 2 ). In the third review, established and novel approaches to manipulate miRNA maturation and activity are described. During the last 5 years many correlations between the levels of certain miRNAs and various human malignancies have been identifi ed, and in some cases the causative role of certain miRNAs in disease formation has been shown. Specifi c miRNAs can regulate certain proteins causing or preventing disease formation. These miRNA–disease correlations call for easy for easy and reliable methods for quantifying specifi c miRNA species from biological samples, a topic which is only partially covered in this book. While miRNAs today have somehow lost their “exotic” touch with many standard methods established in laboratories around the world, novel methods with the potential to expand the view on miRNAs are currently being developed. Such innovation can arise from improving existing methods or by the development of completely new methods allowing for addressing questions other than previous ones. In this book, established methods (qRT-PCR of miRNA maturation components,qRT-PCR of miRNAs) are completed with fl uorescent and nonfl uorescent methods for homogenous assays of Dicer-mediated miRNA maturation or an in vivo assay for Drosha activity. Since miRNAs appear to be emerging drug targets, anti-miRs are already commercially available. Less common is the use of biologically stable PNA as anti-miRs or even miRNA maturation inhibitors, doubtlessly adding to the arsenal of current oligonucleotide approaches to manipulate miRNA activity. Apart from oligonucleotides affecting miRNA activity, which are already under clinical investigation, there is a huge interest in finding small molecules with equivalent effects. Specially adapted luciferase assays have proven as powerful tools for identifying candidate small molecule miRNA effectors. I am convinced that the collection of methods of this book is suitable to widen the view on miRNA as biological mediators and potential drug targets and thus stimulate futurer esearch in this highly dynamic and thrilling topic.

Nucleic Acids Research Methods

Each of the collections on this page is a category-specific archive of Methods published in NAR from 1999 to the present. The numbers in parentheses show the number of articles currently in each collection.

Chapter 1 - Cells and Viruses - Overview
Chapter 2 - Protein Structure and Function
Chapter 3 - Nucleic Acids, Genomics and Proteomics
Chapter 4 - Gene Transcription
Chapter 5 - Posttranscriptional Processes
Chapter 6 - Cell Signaling and Apoptosis
Chapter 7 - DNA Replication, Mutation and Repair
Chapter 8 - Cell Division and DNA Recombination
Chapter 9 - Biotechnology and Bioinformatics
Chapter 10 - Genes and Disease