Jun 2010
RNA Silencing in Plants
RNA Silencing in Plants and the Role of Viral Suppressors
from Ana Giner, Juan Jose Lopez-Moya and Lorant Lakatos writing in RNA Interference and Viruses
The term RNA silencing refers to several pathways present in eukaryotic organisms that lead to the sequence specific elimination or functional blocking of RNAs with homology to double stranded RNAs (dsRNAs) that have previously triggered the mechanism. Besides playing important roles in developmental control, RNA silencing forms part of the defence against viruses in plants, acting as a potent antiviral mechanism. To escape from the RNA silencing-based defence, most plant viruses make use of different strategies, the most common relying in the action of viral proteins with the capacity to suppress RNA silencing. The characterization of these viral suppressors is providing useful insights to understand how RNA silencing works, revealing components and steps in the silencing pathways.
Further reading: Recent Advances in Plant Virology | RNA Interference and Viruses | RNA and the Regulation of Gene Expression
from Ana Giner, Juan Jose Lopez-Moya and Lorant Lakatos writing in RNA Interference and Viruses
The term RNA silencing refers to several pathways present in eukaryotic organisms that lead to the sequence specific elimination or functional blocking of RNAs with homology to double stranded RNAs (dsRNAs) that have previously triggered the mechanism. Besides playing important roles in developmental control, RNA silencing forms part of the defence against viruses in plants, acting as a potent antiviral mechanism. To escape from the RNA silencing-based defence, most plant viruses make use of different strategies, the most common relying in the action of viral proteins with the capacity to suppress RNA silencing. The characterization of these viral suppressors is providing useful insights to understand how RNA silencing works, revealing components and steps in the silencing pathways.
Further reading: Recent Advances in Plant Virology | RNA Interference and Viruses | RNA and the Regulation of Gene Expression
Plant Viral Vectors for Protein Expression
Plant Viral Vectors for Protein Expression
from Yuri Y. Gleba and Anatoli Giritch writing in Recent Advances in Plant Virology
Plant-virus-driven transient expression of heterologous proteins is the basis of several mature manufacturing processes that are currently being used for the production of multiple proteins including vaccine antigens and antibodies. Viral vectors have also become useful tools for research. In recent years, advances have been made both in the development of first-generation vectors (those that employ the 'full virus' strategy) as well as second-generation vectors designed using the 'deconstructed virus' approach. This second strategy relies on Agrobacterium as a vector to deliver DNA copies of one or more viral RNA replicons. Among the most often used viral backbones are those of Tobacco mosaic virus, Potato virus X, and Cowpea mosaic virus. Prototypes of industrial processes that provide for high-yield, rapid scale-up, and fast manufacturing have been recently developed using viral vectors, with several manufacturing facilities compliant with good manufacturing practices (GMP) in place, and a number of pharmaceutical proteins currently in pre-clinical and clinical trials.
Further reading: Recent Advances in Plant Virology | Virology Publications
from Yuri Y. Gleba and Anatoli Giritch writing in Recent Advances in Plant Virology
Plant-virus-driven transient expression of heterologous proteins is the basis of several mature manufacturing processes that are currently being used for the production of multiple proteins including vaccine antigens and antibodies. Viral vectors have also become useful tools for research. In recent years, advances have been made both in the development of first-generation vectors (those that employ the 'full virus' strategy) as well as second-generation vectors designed using the 'deconstructed virus' approach. This second strategy relies on Agrobacterium as a vector to deliver DNA copies of one or more viral RNA replicons. Among the most often used viral backbones are those of Tobacco mosaic virus, Potato virus X, and Cowpea mosaic virus. Prototypes of industrial processes that provide for high-yield, rapid scale-up, and fast manufacturing have been recently developed using viral vectors, with several manufacturing facilities compliant with good manufacturing practices (GMP) in place, and a number of pharmaceutical proteins currently in pre-clinical and clinical trials.
Further reading: Recent Advances in Plant Virology | Virology Publications
RNA Silencing
Category: RNA | Regulation
RNA Silencing and the Interplay Between Plants and Viruses
from Lourdes Fernández-Calvino, Livia Donaire and César Llave writing in Recent Advances in Plant Virology
In eukaryotes, RNA silencing controls gene expression to regulate development, genome stability and stress-induced responses. In plants, this process is also recognized as a major immune system targeted against plant viruses. Plant viruses stimulate RNA silencing responses though formation of viral RNA with double-stranded features that are subsequently processed into functional small RNAs (sRNAs). Recent studies highlight the complexity of the viral sRNA populations and their potential to associate with multiple silencing effector complexes. This fact has profound implications in the cross-talk interactions between plants and viruses since both virus genomes and host genes are putative targets of viral sRNAs. The concept of RNA silencing is an elegant natural antiviral mechanism in plants. Viral sRNA-mediated regulation of gene expression is important in the frame of compatible interactions between plants and viruses.
Further reading: Recent Advances in Plant Virology | Virology Publications | RNA and the Regulation of Gene Expression
from Lourdes Fernández-Calvino, Livia Donaire and César Llave writing in Recent Advances in Plant Virology
In eukaryotes, RNA silencing controls gene expression to regulate development, genome stability and stress-induced responses. In plants, this process is also recognized as a major immune system targeted against plant viruses. Plant viruses stimulate RNA silencing responses though formation of viral RNA with double-stranded features that are subsequently processed into functional small RNAs (sRNAs). Recent studies highlight the complexity of the viral sRNA populations and their potential to associate with multiple silencing effector complexes. This fact has profound implications in the cross-talk interactions between plants and viruses since both virus genomes and host genes are putative targets of viral sRNAs. The concept of RNA silencing is an elegant natural antiviral mechanism in plants. Viral sRNA-mediated regulation of gene expression is important in the frame of compatible interactions between plants and viruses.
Further reading: Recent Advances in Plant Virology | Virology Publications | RNA and the Regulation of Gene Expression
Glycoconjugate Vaccine
Glycoconjugate Vaccine
from David R. Bundle writing in Vaccine Design: Innovative Approaches and Novel Strategies
Methods for single point attachment of polysaccharides and oligosaccharides to protein carriers and T-cell peptides are important in vaccine design. Contemporary approaches involve synthetic oligosaccharides with linker or tether chemistry designed for compatibility with synthetic strategies. Current research involves the synthesis and evaluation of conjugate vaccines designed to combat infectious bacterial and fungal diseases, as well as the design and testing of therapeutic cancer vaccine. The prevailing dogma that protective B-cell epitopes should be comprised of 10-20 monosaccharides is confirmed for several experimental vaccines including those directed toward Shigell flexneri and Shigella dysenteriae. However, several small epitopes composed of 3-5 monosaccharide residues are sufficient to induce antibody against the whole organism and to confer protection.
Further reading: Vaccine Design: Innovative Approaches and Novel Strategies
from David R. Bundle writing in Vaccine Design: Innovative Approaches and Novel Strategies
Methods for single point attachment of polysaccharides and oligosaccharides to protein carriers and T-cell peptides are important in vaccine design. Contemporary approaches involve synthetic oligosaccharides with linker or tether chemistry designed for compatibility with synthetic strategies. Current research involves the synthesis and evaluation of conjugate vaccines designed to combat infectious bacterial and fungal diseases, as well as the design and testing of therapeutic cancer vaccine. The prevailing dogma that protective B-cell epitopes should be comprised of 10-20 monosaccharides is confirmed for several experimental vaccines including those directed toward Shigell flexneri and Shigella dysenteriae. However, several small epitopes composed of 3-5 monosaccharide residues are sufficient to induce antibody against the whole organism and to confer protection.
Further reading: Vaccine Design: Innovative Approaches and Novel Strategies
Genome-derived vaccine
The first genome-derived vaccine now in clinical trials
from Fabio Bagnoli, Nathalie Norais, Ilaria Ferlenghi, Maria Scarselli, Claudio Donati, Silvana Savino, Michèle A. Barocchi and Rino Rappuoli writing in Vaccine Design: Innovative Approaches and Novel Strategies
Genome sequencing has become routine, and modern vaccine design is taking advantage of the accumulating genomic information. Reverse vaccinology is built on genome-based antigen discovery and has largely replaced classical vaccinology methods based on growing and dissecting the microorganism. The main advantage of the approach is the fast prediction of vaccine candidates. Most of the antigens will be surface exposed proteins, since these antigens are most likely accessible to antibodies. This approach can be applied to non-cultivable microorganisms, something difficult or impossible to do with conventional approaches. When the first reverse vaccinology project was started, in the year 2000, antigen identification was mainly based on bioinformatic analysis of one genome. Since then, the technique has shown its full potential, with the first genome-derived vaccine now in clinical trials and several vaccines in preclinical studies. In the meantime the approach has been improved with the support of proteomics, functional genomics and comparative genomics. The complete process includes antigen prediction to high-throughput purification, screening and selection of the vaccine composition.
Further reading: Vaccine Design: Innovative Approaches and Novel Strategies
from Fabio Bagnoli, Nathalie Norais, Ilaria Ferlenghi, Maria Scarselli, Claudio Donati, Silvana Savino, Michèle A. Barocchi and Rino Rappuoli writing in Vaccine Design: Innovative Approaches and Novel Strategies
Genome sequencing has become routine, and modern vaccine design is taking advantage of the accumulating genomic information. Reverse vaccinology is built on genome-based antigen discovery and has largely replaced classical vaccinology methods based on growing and dissecting the microorganism. The main advantage of the approach is the fast prediction of vaccine candidates. Most of the antigens will be surface exposed proteins, since these antigens are most likely accessible to antibodies. This approach can be applied to non-cultivable microorganisms, something difficult or impossible to do with conventional approaches. When the first reverse vaccinology project was started, in the year 2000, antigen identification was mainly based on bioinformatic analysis of one genome. Since then, the technique has shown its full potential, with the first genome-derived vaccine now in clinical trials and several vaccines in preclinical studies. In the meantime the approach has been improved with the support of proteomics, functional genomics and comparative genomics. The complete process includes antigen prediction to high-throughput purification, screening and selection of the vaccine composition.
Further reading: Vaccine Design: Innovative Approaches and Novel Strategies
Conference: Applied Microbiology
Category: Molecular Biology Conference
April 3 - 6, 2011 Annual Conference of the Association for General an Applied Microbiology (VAAM)
Karlsruhe, Germany Further information
Main topics: Cell Biology, Environmental Microbiology, Food Microbiology, Microbial Interactions, New Imaging and other innovative Techniques, Stress Responses, White Biotechnology
Suggested reading: Microbiology Books
Karlsruhe, Germany Further information
Main topics: Cell Biology, Environmental Microbiology, Food Microbiology, Microbial Interactions, New Imaging and other innovative Techniques, Stress Responses, White Biotechnology
Suggested reading: Microbiology Books
Conference: Microbes and Industrial Biotechnology
Category: Molecular Biology Conference
November 21 - 24, 2010 ESF-BU-CeBiTec Conference on Microbes and Industrial Biotechnology
Bielefeld, Germany Further information
Chair: Volker Wendisch, Bielefeld University, Institut fur Genomforschung und Systembiologie, DE, Oluf Kruse, Bielefeld University, Center for Biotechnology. Closing date for application is 10th of September, 2010.
Suggested reading: Microbiology Books
Bielefeld, Germany Further information
Chair: Volker Wendisch, Bielefeld University, Institut fur Genomforschung und Systembiologie, DE, Oluf Kruse, Bielefeld University, Center for Biotechnology. Closing date for application is 10th of September, 2010.
Suggested reading: Microbiology Books
Course: Microscopy, Modeling and Biophysical Methods
September 20 - October 2, 2010 Microscopy, Modeling and Biophysical Methods
Heidelberg, Germany Further information
EMBO Practical Course
Suggested reading: Molecular Biology Books
Heidelberg, Germany Further information
EMBO Practical Course
Suggested reading: Molecular Biology Books
Conference: Protein-Protein Interactions
Category: Molecular Biology Conference
November 14 - 19, 2010 Molecular Perspectives on Protein-Protein Interactions
Sant Feliu de Guixols, Spain Further information
ESF-EMBO Symposium. Chaired by: Dr. Jacob Piehler, University of Osnabruck, DE, Co-Chairs: Gideon Schreiber, Weizmann Institute of Science, IL, Colin Kleanthous, University of York, UK
Suggested reading: Molecular Biology Books
Sant Feliu de Guixols, Spain Further information
ESF-EMBO Symposium. Chaired by: Dr. Jacob Piehler, University of Osnabruck, DE, Co-Chairs: Gideon Schreiber, Weizmann Institute of Science, IL, Colin Kleanthous, University of York, UK
Suggested reading: Molecular Biology Books
Conference: Cytoskeleton from Molecules to Cells
Category: Molecular Biology Conference
October 3 - 8, 2010 Emergent Properties of the Cytoskeleton: Molecules to Cells
Sant Feliu de Guixols, Spain Further information
Chaired by: Michelle Peckham, University of Leeds, Institute of Molecular and Cellular Biology, Centre for Human Biology, UK, Claudia Veigel, National Institute of Medical Research, Physical Biochemistry Department, UK
Suggested reading: Molecular Biology Books
Sant Feliu de Guixols, Spain Further information
Chaired by: Michelle Peckham, University of Leeds, Institute of Molecular and Cellular Biology, Centre for Human Biology, UK, Claudia Veigel, National Institute of Medical Research, Physical Biochemistry Department, UK
Suggested reading: Molecular Biology Books
Conference: Transcription and Chromatin
Category: Molecular Biology Conference
August 28 - 30, 2010 Transcription and Chromatin
Heidelberg, Germany Further information
9th EMBL Conference. Transcription and Chromatin. EMBL Courses and Conferences. Understanding the complexity and functional composition of cellular and synaptic networks in the nervous system is a major challenge in neurobiology. Genes and molecules impact directly the assembly, function, and plasticity of specific neural circuits, and recent studies in different model systems start to elucidate the functionality of neuronal connectomes as an higher organisational entity required for the generation of complex behaviours. The goal of this Symposium is to highlight recent work on the anatomical and functional analysis of behaviourally-relevant neural circuits in genetically tractable model systems, and to promote the exchange of ideas and methods in this exciting field of research.
Suggested reading: Molecular Biology Books
Heidelberg, Germany Further information
9th EMBL Conference. Transcription and Chromatin. EMBL Courses and Conferences. Understanding the complexity and functional composition of cellular and synaptic networks in the nervous system is a major challenge in neurobiology. Genes and molecules impact directly the assembly, function, and plasticity of specific neural circuits, and recent studies in different model systems start to elucidate the functionality of neuronal connectomes as an higher organisational entity required for the generation of complex behaviours. The goal of this Symposium is to highlight recent work on the anatomical and functional analysis of behaviourally-relevant neural circuits in genetically tractable model systems, and to promote the exchange of ideas and methods in this exciting field of research.
Suggested reading: Molecular Biology Books
ABC Transporters Book Review
Category: Books | Book Review
I am pleased to provide the following excerpt from a book review of ABC Transporters in Microorganisms:
"of practical use to any scientist working on active transport systems whether in bacteria, parasites, or human cells. It is written in a fashion that allows readers to focus on specific topics and shows comparisons between systems. All the authors are from different disciplines but have contributed their knowledge to a cohesive book ... The book contains some excellent figures of the folding patterns of the proteins and the dynamics of how they change to import or export specific substrates ... well-organized and well-written book ... should be considered an essential reference for laboratories working in this area." from Rebecca T. Horvat (University of Kansas Medical Center, USA) writing in Doodys read more ...
"of practical use to any scientist working on active transport systems whether in bacteria, parasites, or human cells. It is written in a fashion that allows readers to focus on specific topics and shows comparisons between systems. All the authors are from different disciplines but have contributed their knowledge to a cohesive book ... The book contains some excellent figures of the folding patterns of the proteins and the dynamics of how they change to import or export specific substrates ... well-organized and well-written book ... should be considered an essential reference for laboratories working in this area." from Rebecca T. Horvat (University of Kansas Medical Center, USA) writing in Doodys read more ...
 | Edited by: Alicia Ponte-Sucre "well-organized and well-written ... an essential reference" (Doodys)ISBN: 978-1-904455-49-3 Publisher: Caister Academic Press Publication Date: August 2009 Cover: hardback |
Small RNAs
Category: Technology | RNA
The small RNAs of Salmonella
from Sridhar Javayel, Kai Papenfort and Jörg Vogel writing in Salmonella: From Genome to Function
To date, close to one hundred distinct small noncoding RNAs (sRNAs) have been identified in Salmonella by a variety of biocomputational or wet-lab approaches including RNA sequencing. The function of more than twenty of these sRNAs is known from studies in Salmonella itself or can be inferred from conserved homologs in E. coli Many of these sRNAs act in conjunction with the RNA-chaperone Hfq to post-transcriptionally repress or activate trans-encoded target genes, but cis-antisense RNAs and regulators of protein activity are also abundantly present. In addition to a large number of sRNAs conserved in other enteric bacteria, Salmonella also expresses a set of sRNAs specific to this genus. Interestingly, such regulators have been shown to control the expression of conserved genes encoded on the "core" Salmonella genome. Conversely, conserved sRNA can act as regulators of recently acquired Salmonella-specific genes, indicating significant cross-talk of conserved and horizontally acquired elements at the RNA level. A recent review covers strategies for the identification of sRNAs as well as their characterized functional roles in Salmonella.
Further reading: Salmonella: From Genome to Function | RNA and the Regulation of Gene Expression
from Sridhar Javayel, Kai Papenfort and Jörg Vogel writing in Salmonella: From Genome to Function
To date, close to one hundred distinct small noncoding RNAs (sRNAs) have been identified in Salmonella by a variety of biocomputational or wet-lab approaches including RNA sequencing. The function of more than twenty of these sRNAs is known from studies in Salmonella itself or can be inferred from conserved homologs in E. coli Many of these sRNAs act in conjunction with the RNA-chaperone Hfq to post-transcriptionally repress or activate trans-encoded target genes, but cis-antisense RNAs and regulators of protein activity are also abundantly present. In addition to a large number of sRNAs conserved in other enteric bacteria, Salmonella also expresses a set of sRNAs specific to this genus. Interestingly, such regulators have been shown to control the expression of conserved genes encoded on the "core" Salmonella genome. Conversely, conserved sRNA can act as regulators of recently acquired Salmonella-specific genes, indicating significant cross-talk of conserved and horizontally acquired elements at the RNA level. A recent review covers strategies for the identification of sRNAs as well as their characterized functional roles in Salmonella.
Further reading: Salmonella: From Genome to Function | RNA and the Regulation of Gene Expression
High-throughput analysis
Category: Technology
High-throughput screening to determine the genetic requirements for Salmonella survival under different growth conditions
from Mollie Megan Reynolds, Rocio Canals, Michael McClelland and Helene Andrews-Polymenis writing in Salmonella: From Genome to Function
Salmonella species are capable of survival in a wide range of niches, both in the environment and in an infected host. Genetic requirements for survival of Salmonella in different niches have traditionally been identified using gene expression and forward genetics. The availability of complete genome sequences, microarray technology, and cost-effective new sequencing capabilities enabled increasingly efficient high-throughput analyses of Salmonella genomes to identify elements that contribute to survival in these niches. A recent review describes many of the high-throughput tools that have been developed over the past two decades, and the genetic requirements for Salmonella survival that have been identified using these techniques.
Further reading: Salmonella: From Genome to Function
from Mollie Megan Reynolds, Rocio Canals, Michael McClelland and Helene Andrews-Polymenis writing in Salmonella: From Genome to Function
Salmonella species are capable of survival in a wide range of niches, both in the environment and in an infected host. Genetic requirements for survival of Salmonella in different niches have traditionally been identified using gene expression and forward genetics. The availability of complete genome sequences, microarray technology, and cost-effective new sequencing capabilities enabled increasingly efficient high-throughput analyses of Salmonella genomes to identify elements that contribute to survival in these niches. A recent review describes many of the high-throughput tools that have been developed over the past two decades, and the genetic requirements for Salmonella survival that have been identified using these techniques.
Further reading: Salmonella: From Genome to Function
Microfluidic Emulsion PCR
Microfluidic Emulsion PCR
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 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
PCR High Resolution Melting Analysis
High Resolution Melting Analysis
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.
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.
Further reading: PCR Troubleshooting and Optimization: The Essential Guide
PCR in Epigenetics
PCR Applications for Epigenetics Research
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.
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.
Further reading: PCR Troubleshooting and Optimization: The Essential Guide
PCR: MIQE
The MIQE Guidelines Uncloaked
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. An expanded explanation of the guideline items on how those requirements might be met should be consulted prior to publication.
Further reading: PCR Troubleshooting and Optimization: The Essential Guide
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. An expanded explanation of the guideline items on how those requirements might be met should be consulted prior to publication.
Further reading: PCR Troubleshooting and Optimization: The Essential Guide
PCR Data Analysis
qPCR Data Analysis: Unlocking the Secret to Successful Results
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. Data analysis aspects include 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 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. Data analysis aspects include 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
PCR Instrumentation
Real-Time PCR Instrumentation: An Instrument Selection 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 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. 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). Key features differentiate Real-Time PCR instruments, and various criteria should be considered when selecting the instrument that best fits a specific scientist's research needs.
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 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. 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). Key features differentiate Real-Time PCR instruments, and various criteria should be considered when selecting the instrument that best fits a specific scientist's research needs.
Further reading: PCR Troubleshooting and Optimization: The Essential Guide
PCR Optimization
RT-PCR Optimization Strategies
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. Optimization of the whole RT-PCR workflow is important and recommendations to reduce experimental variance and produce more reproducible and reliable results should be followed.
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. Optimization of the whole RT-PCR workflow is important and recommendations to reduce experimental variance and produce more reproducible and reliable results should be followed.
Further reading: PCR Troubleshooting and Optimization: The Essential Guide
PCR Sensitivity
Obtaining Maximum PCR Sensitivity and Specificity
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. Addressing all of these factors will aid the investigator in designing high quality PCR assays.
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. Addressing all of these factors will aid the investigator in designing high quality PCR assays.
Further reading: PCR Troubleshooting and Optimization: The Essential Guide
PCR Controls and Standards
Significance of Controls and Standard Curves in PCR
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. There are strategies for preparing both negative and positive controls for PCR, when they should be employed and how to interpret the information they provide. Standard curves are vital for determining the initial starting amount of the target template and for assessing assay efficiency, precision, sensitivity, and dynamic range. It is important to know how to prepare standards, interpret standard curve and troubleshoot inefficient qPCR reactions.
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. There are strategies for preparing both negative and positive controls for PCR, when they should be employed and how to interpret the information they provide. Standard curves are vital for determining the initial starting amount of the target template and for assessing assay efficiency, precision, sensitivity, and dynamic range. It is important to know how to prepare standards, interpret standard curve and troubleshoot inefficient qPCR reactions.
Further reading: PCR Troubleshooting and Optimization: The Essential Guide
PCR Inhibitors
Difficult Templates and Inhibitors of PCR
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, there are strategies for detecting, avoiding and/or eliminating inhibition during reverse transcription, PCR, RT-PCR and qPCR. Also there are strategies to amplify difficult templates and optimize reverse transcription reactions.
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, there are strategies for detecting, avoiding and/or eliminating inhibition during reverse transcription, PCR, RT-PCR and qPCR. Also there are strategies to amplify difficult templates and optimize reverse transcription reactions.
Further reading: PCR Troubleshooting and Optimization: The Essential Guide
PCR: A Brief History
Category: PCR
A Brief History of PCR
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. A recent review by Wittwer and Farrar discusses the origins of PCR, its early evolution 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. 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.
Read more: 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. A recent review by Wittwer and Farrar discusses the origins of PCR, its early evolution 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. 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.
Read more: PCR Troubleshooting and Optimization: The Essential Guide
Conference Alert: Non-Coding Genome
Category: Conferences | RNA
October 13 - 16, 2010 The Non-Coding Genome
Heidelberg, Germany Further information
This symposium will provide an interdisciplinary discussion of the roles of non-coding RNAs with the aim of enhancing our understanding of gene regulation and function. Topics will include recent discoveries in the fields of prokaryotic and eukaryotic long and short non-coding RNAs. The functional roles of non-coding RNAs in a wide variety of cell processes will be discussed.
Suggested reading: RNA Interference and Viruses: Current Innovations and Future Trends
Heidelberg, Germany Further information
This symposium will provide an interdisciplinary discussion of the roles of non-coding RNAs with the aim of enhancing our understanding of gene regulation and function. Topics will include recent discoveries in the fields of prokaryotic and eukaryotic long and short non-coding RNAs. The functional roles of non-coding RNAs in a wide variety of cell processes will be discussed.
Suggested reading: RNA Interference and Viruses: Current Innovations and Future Trends
Conference alert: Neural Circuits
Category: Conferences
September 5 - 8, 2010 Structure and Function of Neural Circuits
Heidelberg, Germany Further information
Understanding the complexity and functional composition of cellular and synaptic networks in the nervous system is a major challenge in neurobiology. Genes and molecules impact directly the assembly, function, and plasticity of specific neural circuits, and recent studies in different model systems start to elucidate the functionality of neuronal connectomes as an higher organisational entity required for the generation of complex behaviours. The goal of this Symposium is to highlight recent work on the anatomical and functional analysis of behaviourally-relevant neural circuits in genetically tractable model systems, and to promote the exchange of ideas and methods in this exciting field of research.
Suggested reading: Molecular Biology Books
Heidelberg, Germany Further information
Understanding the complexity and functional composition of cellular and synaptic networks in the nervous system is a major challenge in neurobiology. Genes and molecules impact directly the assembly, function, and plasticity of specific neural circuits, and recent studies in different model systems start to elucidate the functionality of neuronal connectomes as an higher organisational entity required for the generation of complex behaviours. The goal of this Symposium is to highlight recent work on the anatomical and functional analysis of behaviourally-relevant neural circuits in genetically tractable model systems, and to promote the exchange of ideas and methods in this exciting field of research.
Suggested reading: Molecular Biology Books
PCR Seminar Online
No matter how good you are at PCR, you can always learn something from the speakers we have lined up for our Getting the most out of PCR live online seminar series. These guy eat, sleep and drink PCR.
Next up we have MIQE Guidelines Uncloaked, in which Greg Shipley will give you the inside track on the requirements you need to satisfy to make sure your PCR results are suitable for publication. You'd be mad to miss it.
This event goes out live tomorrow (Tue 8th June) at 9am Pacific / 12pm Eastern / 5pm BST (UK) / 6pm CET. Click here to secure one of the remaining places on this live event..
You can also click here to take a look at our archive for this series, which now contains:
Magic in Solution: An Introduction and Brief History of PCR
Speaker: Carl Wittwer
Obtaining Maximum PCR Sensitivity and Specificity
Speaker: Cameron N. Gundry Attendence: 125
Significance of Controls and Standard Curves in PCR
Speaker: Ian Kavanagh
Next up we have MIQE Guidelines Uncloaked, in which Greg Shipley will give you the inside track on the requirements you need to satisfy to make sure your PCR results are suitable for publication. You'd be mad to miss it.
This event goes out live tomorrow (Tue 8th June) at 9am Pacific / 12pm Eastern / 5pm BST (UK) / 6pm CET. Click here to secure one of the remaining places on this live event..
You can also click here to take a look at our archive for this series, which now contains:
Magic in Solution: An Introduction and Brief History of PCR
Speaker: Carl Wittwer
Obtaining Maximum PCR Sensitivity and Specificity
Speaker: Cameron N. Gundry Attendence: 125
Significance of Controls and Standard Curves in PCR
Speaker: Ian Kavanagh
Streptomyces book
Paul Dyson (Institute of Life Sciences, School of Medicine, Swansea, UK) presents a new book on Streptomyces: Molecular Biology and Biotechnology
Streptomycetes are Gram-positive, high GC-content, sporulating bacteria found predominantly in soil. Streptomycetes are characterised by a complex secondary metabolism producing antibiotic compounds and other metabolites with medicinal properties. In recent years genomic studies, genomic mining and biotechnological approaches have been employed in the search for new antibiotics and other drugs.
With contributions from some of the leading scientists in the field, this volume documents recent research and development in streptomycetes genomics, physiology and metabolism. With a focus on biotechnology and genomics, the book provides an excellent source of up-to-date information. Topics include: genome architecture, conjugative genetic elements, differentiation, protein secretion, central carbon metabolic pathways, regulation of nitrogen assimilation, phosphate control of metabolism, gamma-butyrolactones and their role in antibiotic regulation, clavulanic acid and clavams, genome-guided exploration, gene clusters for bioactive natural products, genomics of cytochromes p450.
Streptomycetes are Gram-positive, high GC-content, sporulating bacteria found predominantly in soil. Streptomycetes are characterised by a complex secondary metabolism producing antibiotic compounds and other metabolites with medicinal properties. In recent years genomic studies, genomic mining and biotechnological approaches have been employed in the search for new antibiotics and other drugs.
With contributions from some of the leading scientists in the field, this volume documents recent research and development in streptomycetes genomics, physiology and metabolism. With a focus on biotechnology and genomics, the book provides an excellent source of up-to-date information. Topics include: genome architecture, conjugative genetic elements, differentiation, protein secretion, central carbon metabolic pathways, regulation of nitrogen assimilation, phosphate control of metabolism, gamma-butyrolactones and their role in antibiotic regulation, clavulanic acid and clavams, genome-guided exploration, gene clusters for bioactive natural products, genomics of cytochromes p450.
 | Edited by: Paul Dyson ISBN: 978-1-904455-77-6 Publisher: Caister Academic Press Publication Date: January 2011 Cover: hardback |
Essential reading for research scientists, biotechnologists, graduate students and other professionals involved in streptomycetes research, antibiotic and antimicrobial development, drug discovery, soil microbiology and related fields. A recommended text for all microbiology laboratories.