This document summarizes a short course on DNA barcoding methods that will cover equipment needed for PCR amplification, PCR protocols, product verification using agarose gels, minimizing contamination risks, and shipping and storing DNA extracts. The course will provide an overview of typical PCR protocols and note that protocols depend on sample quality and scale of operations. Small PCR volumes and use of trehalose are discussed as cost-saving measures. Primer selection is also highlighted as a key factor in success.
Reproducibility, Quality Control and Importance of AutomationQIAGEN
In this webinar, we will introduce you to the key sample quality parameters, discuss their respective impact on downstream applications and how to monitor them, and present the advantages of automating quality control along complex workflows.
Back to basics: Fundamental Concepts and Special Considerations in RNA IsolationQIAGEN
RNA integrity and quality are critical to obtain meaningful and reliable downstream data. This slidedeck details the challenges and considerations of handling RNA samples, RNA stabilization, the need for quality control analysis and common methods for RNA integrity and quality assessment.
Process development guidance for AAV and lentivirus manufacturing based on co...MilliporeSigma
Access the interactive recording here: https://bit.ly/37nl3Ex
Webinar summary:
An efficient production platform is essential for successful commercial implementation of gene therapy programs. AAV and Lentivirus manufacturing process are often developed with compressed timelines, reduced process optimization and low product yields which can have significant effect on costs.
In this webinar, you will learn:
* How manufacturing costs are examined for adeno-associated virus and lentivirus production with several different for each vector
* That key process characteristics like production titer, production of empty viral particles, downstream product recovery, and the batching strategy can effect the overall manufacturing cost
* How holistic evaluation is an important tool during process development to help prioritize different approaches to improve viral vector production processes
Abstract:
An efficient production platform is essential for successful commercial implementation of gene therapy programs. Viral vector manufacturing processes are often developed under timelines which are considerably shorter than those for more mature biopharmaceuticals. Consequently, the level of process optimization is reduced and challenges related to low product yields are common. These factors, as well as the small batch sizes common for these processes, can have significant effect on manufacturing costs.
RNA integrity and quality - Standardize RNA quality controlQIAGEN
RNA integrity and quality are critical to obtain meaningful and reliable downstream data. This slidedeck details the important considerations and critical factors in RNA preparation. It also highlights the need for quality control analysis and common methods for RNA integrity and quality assessment.
Practical hints and new solutions for successful real-time PCR studies QIAGEN
Part 1: Practical hints and new solutions for successful real-time PCR studies
In this webinar we will cover the following topics which are critical steps for efficient and precise gene expression studies using real-time PCR technology:
- Effect of RNA integrity on real-time PCR results – tips to achieve a true RNA profiling suitable for real-time PCR studies
- Improved methods for cDNA synthesis, optimized for real-time PCR
- Real-time PCR analysis
o Real-time PCR essentials and background information on different quantification strategies
o SYBR Green real-time PCR – factors influencing specificity
o Introduction to probe technology
o New, fast and efficient real-time PCR solutions
Part 2: Critical Factors for Successful Multiplex Real-Time PCR
Multiplex real-time PCR is a powerful tool for gene expression analysis, viral load monitoring, genotyping, and many other applications. The ability to amplify and detect several genomic DNA, cDNA, or RNA targets in the same reaction offers many benefits:
• Conservation of precious samples – more quantification data per sample
• Increased throughput – more targets analyzed per run on a cycler
• Reliable results – no well-to-well variability due to co-amplification of internal control
• Reduced costs – save time and reagents
The QuantiFast Multiplex PCR and RT-PCR kits are optimized for reliable amplification of many different templates despite a high variability in abundance. Thus they enable successful amplification of multiple targets on the first attempt without optimization.
This webinar explains the principles of the QIAGEN multiplex technologies and shows data demonstrating the exceptional multiplex real-time PCR performance of the QuantiFast Multiplex kits.
In this slidedeck, the following topics, which are critical steps for efficient and precise gene expression studies using real-time PCR technology, are covered:
• Effect of RNA integrity on real-time PCR results – tips on how to achieve a true RNA profile suitable for real-time PCR studies
• Improved methods for cDNA synthesis, optimized for real-time PCR
• Real-time PCR analysis
• Real-time PCR essentials and background information on different quantification strategies
• SYBR Green real-time PCR – factors influencing specificity
• Introduction to probe technology
• New, fast and efficient real-time PCR solutions
Advancing Microbiome Research: From challenging samples to insight with Confi...QIAGEN
Microbiome research encompasses sample types as diverse as the human gut, Antarctic soil, ocean water and acidic hot spring biofilms. These samples are challenging because they are difficult to lyse, with some microbes containing a tough extracellular matrix. Incomplete lysis of a microbial community results in an inaccurate representation of the microbial content of the sample. Additionally, PCR inhibitors present in these samples, especially humic acids, polysaccharides, polyphenolics, lipids and heavy metals result in inaccurate quantification of nucleic acids that may inhibit downstream applications such as qPCR and NGS.
Semi Automated Low-throughput Workflow for Microbial Analyses of Human StoolQIAGEN
The gut microbiota composition changes dramatically throughout aging and disease. A healthy gut microbiota is typically characterized by large bacterial taxonomic diversity and functional capacity, whereas frailty and aging are associated with loss of diversity and expansion of more pathogenic bacterial species. However, in order to accurately profile changes in microbial communities, the reproducible isolation of high-quality DNA is an important step. Automation enables reliable and reproducible isolation of DNA of superior quality, which can be used directly for downstream sequencing applications.
This webinar focuses on the development of a semi-automated workflow to profile the gut microbiota of young and old individuals and identify changes in bacterial composition and function that occur with age. This workflow will help to simplify and streamline the DNA extraction process for samples with high inhibitor content and subsequent microbial community analyses.
Reproducibility, Quality Control and Importance of AutomationQIAGEN
In this webinar, we will introduce you to the key sample quality parameters, discuss their respective impact on downstream applications and how to monitor them, and present the advantages of automating quality control along complex workflows.
Back to basics: Fundamental Concepts and Special Considerations in RNA IsolationQIAGEN
RNA integrity and quality are critical to obtain meaningful and reliable downstream data. This slidedeck details the challenges and considerations of handling RNA samples, RNA stabilization, the need for quality control analysis and common methods for RNA integrity and quality assessment.
Process development guidance for AAV and lentivirus manufacturing based on co...MilliporeSigma
Access the interactive recording here: https://bit.ly/37nl3Ex
Webinar summary:
An efficient production platform is essential for successful commercial implementation of gene therapy programs. AAV and Lentivirus manufacturing process are often developed with compressed timelines, reduced process optimization and low product yields which can have significant effect on costs.
In this webinar, you will learn:
* How manufacturing costs are examined for adeno-associated virus and lentivirus production with several different for each vector
* That key process characteristics like production titer, production of empty viral particles, downstream product recovery, and the batching strategy can effect the overall manufacturing cost
* How holistic evaluation is an important tool during process development to help prioritize different approaches to improve viral vector production processes
Abstract:
An efficient production platform is essential for successful commercial implementation of gene therapy programs. Viral vector manufacturing processes are often developed under timelines which are considerably shorter than those for more mature biopharmaceuticals. Consequently, the level of process optimization is reduced and challenges related to low product yields are common. These factors, as well as the small batch sizes common for these processes, can have significant effect on manufacturing costs.
RNA integrity and quality - Standardize RNA quality controlQIAGEN
RNA integrity and quality are critical to obtain meaningful and reliable downstream data. This slidedeck details the important considerations and critical factors in RNA preparation. It also highlights the need for quality control analysis and common methods for RNA integrity and quality assessment.
Practical hints and new solutions for successful real-time PCR studies QIAGEN
Part 1: Practical hints and new solutions for successful real-time PCR studies
In this webinar we will cover the following topics which are critical steps for efficient and precise gene expression studies using real-time PCR technology:
- Effect of RNA integrity on real-time PCR results – tips to achieve a true RNA profiling suitable for real-time PCR studies
- Improved methods for cDNA synthesis, optimized for real-time PCR
- Real-time PCR analysis
o Real-time PCR essentials and background information on different quantification strategies
o SYBR Green real-time PCR – factors influencing specificity
o Introduction to probe technology
o New, fast and efficient real-time PCR solutions
Part 2: Critical Factors for Successful Multiplex Real-Time PCR
Multiplex real-time PCR is a powerful tool for gene expression analysis, viral load monitoring, genotyping, and many other applications. The ability to amplify and detect several genomic DNA, cDNA, or RNA targets in the same reaction offers many benefits:
• Conservation of precious samples – more quantification data per sample
• Increased throughput – more targets analyzed per run on a cycler
• Reliable results – no well-to-well variability due to co-amplification of internal control
• Reduced costs – save time and reagents
The QuantiFast Multiplex PCR and RT-PCR kits are optimized for reliable amplification of many different templates despite a high variability in abundance. Thus they enable successful amplification of multiple targets on the first attempt without optimization.
This webinar explains the principles of the QIAGEN multiplex technologies and shows data demonstrating the exceptional multiplex real-time PCR performance of the QuantiFast Multiplex kits.
In this slidedeck, the following topics, which are critical steps for efficient and precise gene expression studies using real-time PCR technology, are covered:
• Effect of RNA integrity on real-time PCR results – tips on how to achieve a true RNA profile suitable for real-time PCR studies
• Improved methods for cDNA synthesis, optimized for real-time PCR
• Real-time PCR analysis
• Real-time PCR essentials and background information on different quantification strategies
• SYBR Green real-time PCR – factors influencing specificity
• Introduction to probe technology
• New, fast and efficient real-time PCR solutions
Advancing Microbiome Research: From challenging samples to insight with Confi...QIAGEN
Microbiome research encompasses sample types as diverse as the human gut, Antarctic soil, ocean water and acidic hot spring biofilms. These samples are challenging because they are difficult to lyse, with some microbes containing a tough extracellular matrix. Incomplete lysis of a microbial community results in an inaccurate representation of the microbial content of the sample. Additionally, PCR inhibitors present in these samples, especially humic acids, polysaccharides, polyphenolics, lipids and heavy metals result in inaccurate quantification of nucleic acids that may inhibit downstream applications such as qPCR and NGS.
Semi Automated Low-throughput Workflow for Microbial Analyses of Human StoolQIAGEN
The gut microbiota composition changes dramatically throughout aging and disease. A healthy gut microbiota is typically characterized by large bacterial taxonomic diversity and functional capacity, whereas frailty and aging are associated with loss of diversity and expansion of more pathogenic bacterial species. However, in order to accurately profile changes in microbial communities, the reproducible isolation of high-quality DNA is an important step. Automation enables reliable and reproducible isolation of DNA of superior quality, which can be used directly for downstream sequencing applications.
This webinar focuses on the development of a semi-automated workflow to profile the gut microbiota of young and old individuals and identify changes in bacterial composition and function that occur with age. This workflow will help to simplify and streamline the DNA extraction process for samples with high inhibitor content and subsequent microbial community analyses.
Cancer Research & the Challenges of FFPE Samples – An IntroductionQIAGEN
A cascade of complex genetic and epigenetic changes regulate tumor formation and progression. Gene expression analyses can shed light on these changes at a molecular level and identify the key genes and associated pathways involved in cancer. Often the samples used in cancer research are FFPE samples, which pose a significant challenge in terms of nucleic acid quality. The quality of nucleic acids extracted from FFPE samples depends on a number of factors, including how the samples were handled before, during and after fixation and embedding.
Dr. Vishwadeepak Tripathi describes the variability of sample purification from FFPE samples – in particular, samples to be used in cancer research. What are the challenges and solutions, and what quality control approach can ensure credible results? This webinar will focus on sample purification and the quality control of FFPE samples and compare different automated purification procedures.
RotorGene Q A Rapid, Automatable real-time PCR Instrument for Genotyping and...QIAGEN
QIAGEN has developed a selection of robust, novel chemistries to prevent PCR crosstalk. We can successfully measure target abundance and fold change in real-time assays, and perform sub-genotyping using a fast, high-throughput and powerful High-Resolution Melting (HRM) statistical analysis program. In this presentation, we will demonstrate these features and benefits with examples.
RNA Integrity and Quality – Standardize RNA Quality Control QIAGEN
RNA integrity and quality are critical to obtain meaningful and reliable downstream data. This slidedeck details the challenges and considerations of handling RNA samples, the need for quality control analysis and common methods for RNA integrity and quality assessment. The QIAxcel Advanced System will be introduced to automate the process of RNA sample integrity analysis and obtain objective quality measurement. Application data will be presented.
Critical Steps for Real-Time PCR Analysis: Tips and Solutions to Achieve Effi...QIAGEN
In this slidedeck, we cover the following topics which are critical steps for efficient and precise gene expression studies using real-time PCR technology:
1) Effect of RNA integrity on real-time PCR results – tips to achieve a true RNA profiling suitable for real-time PCR studies
2) Improved methods for cDNA synthesis, optimized for real-time PCR
3) Real-time PCR analysis:
• Real-time PCR essentials and background information on different quantification strategies
• SYBR Green real-time PCR – factors influencing specificity
• Introduction to probe technology
• New, fast and efficient real-time PCR solutions
Nucleic Acid Quantification from FFPE Samples – Are You Doing it Right?QIAGEN
Formalin-fixation and paraffin-embedding is a standard method for long-term preservation of tissue biopsies and these stored samples are a valuable tool for studying diseases such as cancer, especially when they are histologically and pathologically well characterized, and follow-up clinical data is available. The quality of nucleic acids extracted from FFPE samples is influenced by a number of factors, including how the samples were handled before, during and after fixation and embedding. Moreover, there are several difficulties when purifying nucleic acids from FFPE samples as the chemicals and temperature used during the process can degrade the DNA.
In this webinar, we will discuss the variability in quantity and purity of DNA purified from FFPE material. We will show data from different quantification and quality control methods, demonstrate the impact of inaccurate quantification on downstream results and discuss how to overcome these challenges.
Webinar: Evaluating Viral Clearance for Continuous ProcessesMilliporeSigma
Participate in the interactive webinar now: http://bit.ly/ViralClearanceWebinar
Is viral clearance a hurdle to implementing continuous processing? We’ll share virus spiking alternatives that may pave the way for effectively evaluating viral clearance by chromatography steps in a continuous process.
Explore our webinar library: www.emdmillipore.com/webinars
Optimal RNAlater® incubation and removal conditions prior to isolation of tot...QIAGEN
RNA is highly sensitive to degradation. Handling methods and prolonged storage of cells can greatly affect the quality of the RNA that can be later isolated. Contamination with RNases is the most significant problem, especially as they are so ubiquitous in the environment. They can degrade RNA to the point where results of downstream analyses become meaningless.
Submerging cells in RNAlater, an RNA stabilization reagent, helps to stabilize the RNA within the cells and prevent degradation, supporting accurate downstream gene expression analyses. However, to avoid any interference from any RNAlater components in isolation and analyses, cells must be pelleted and the reagent must be removed. The separation of cells from excess RNAlater via centrifugation is impeded due to the higher density of the reagent compared to standard culture medium. This means it requires higher centrifugal forces, which might damage cells due to increased shearing forces, leading to reduced RNA yield. The aim of this study was to establish the optimal conditions for the recovery of cells from RNAlater after RNA stabilization for maximum RNA yield and integrity.
Process Development for Cell Therapy and Viral Gene TherapyMerck Life Sciences
Today’s viral vector manufacturing processes remain challenging. Process development is a critical enabler to bring safe, effective, sustainable products to market to address patient needs. When done properly, it can reduce the timeline of the project and the cost of producing the therapeutic product.
The webinar discusses our strategies for developing lentivirus and adeno associated virus (AAV) and the impact these early decisions can have on commercial readiness.
Watch the interactive webinar now: https://bit.ly/2VplwQq
PCR Array Data Analysis Tutorial: qPCR Technology Webinar Series Part 3QIAGEN
Using actual PCR Array data, this slidedeck presents an easy-to-use and free web-based data analysis tool to calculate fold-differences in gene expression from your raw real-time PCR threshold cycles. Learn how you can look at your results in different formats, including heat map, scatter, volcano, clustergram and multigroup plot.
Stable 16 year storage of DNA purified with the QIAamp® DNA Blood mini kit - ...QIAGEN
In this application note, we describe the success of the QIAamp DNA Blood Mini Kit in the preparation of highly stable DNA,as evidenced by 16-year storage data. We also report the best storage conditions for maximal protection against degradation.
Simultaneous Isolation of RNA & DNA from one FFPE SampleQIAGEN
Worldwide, there are millions of tissue samples archived in tissue biobanks and biorepositories. These samples are extremely valuable for pharmacological and biomedical research and companion diagnostics, due to the linkage to patient history. The vast majority of archived tissue samples are formalin-fixed and paraffin-embedded (FFPE), since formalin is the standard fixative for tissue samples.
FFPE blocks serve as an excellent source for histomorphology studies, but their use in molecular studies is challenging, due to crosslinking and fragmentation caused by fixation, processing, embedding, and storage conditions. For reliable comparison of genomic and transcriptomic data from heterogeneous samples and to spare sample material, purification of DNA and RNA from
the same sample is essential. This is particularly important when working with tumorous tissues, which contain a heterogeneous distribution of healthy and malignant cells.
Liquid biopsy: Overcome Challenges of Circulating DNA with Automated and Stan...QIAGEN
Circulating cell-free DNA (ccfDNA) originating from malignant tumors, a developing fetus and also from inflammatory tissues, is present in the cell-free nucleic acids in plasma, serum and other body fluids and is considered a “liquid biopsy”. Access to ccfDNA for analysis allows for specific detection of certain disease states based on a simple blood sample. Circulating cell-free DNA shows distinctive properties – it is present mostly as shorter fragments of less than 500 bp and the concentration of ccfDNA in a plasma or serum sample is low (approximately 1–100 ng/ml) compared to cellular materials and varies considerably between different individuals.
Because of their fragmented nature and low concentration, ccfDNA presents a particular challenge for efficient extraction / purification and quantification, such as by qPCR. We present data on solutions for the following critical problems concerning the purification of ccfDNA for research and molecular diagnostic applications:
• Pre-analytical workflow (blood processing) for analyzing ccfDNA
• Optimization of ccfDNA extraction from plasma samples: low target concentrations require efficient ccfDNA enrichment from larger sample volumes
• Novel automated extraction of ccfDNA using the QIAsymphony SP instrument for liquid biopsy diagnostic applications.
Cancer Research & the Challenges of FFPE Samples – An IntroductionQIAGEN
A cascade of complex genetic and epigenetic changes regulate tumor formation and progression. Gene expression analyses can shed light on these changes at a molecular level and identify the key genes and associated pathways involved in cancer. Often the samples used in cancer research are FFPE samples, which pose a significant challenge in terms of nucleic acid quality. The quality of nucleic acids extracted from FFPE samples depends on a number of factors, including how the samples were handled before, during and after fixation and embedding.
Dr. Vishwadeepak Tripathi describes the variability of sample purification from FFPE samples – in particular, samples to be used in cancer research. What are the challenges and solutions, and what quality control approach can ensure credible results? This webinar will focus on sample purification and the quality control of FFPE samples and compare different automated purification procedures.
RotorGene Q A Rapid, Automatable real-time PCR Instrument for Genotyping and...QIAGEN
QIAGEN has developed a selection of robust, novel chemistries to prevent PCR crosstalk. We can successfully measure target abundance and fold change in real-time assays, and perform sub-genotyping using a fast, high-throughput and powerful High-Resolution Melting (HRM) statistical analysis program. In this presentation, we will demonstrate these features and benefits with examples.
RNA Integrity and Quality – Standardize RNA Quality Control QIAGEN
RNA integrity and quality are critical to obtain meaningful and reliable downstream data. This slidedeck details the challenges and considerations of handling RNA samples, the need for quality control analysis and common methods for RNA integrity and quality assessment. The QIAxcel Advanced System will be introduced to automate the process of RNA sample integrity analysis and obtain objective quality measurement. Application data will be presented.
Critical Steps for Real-Time PCR Analysis: Tips and Solutions to Achieve Effi...QIAGEN
In this slidedeck, we cover the following topics which are critical steps for efficient and precise gene expression studies using real-time PCR technology:
1) Effect of RNA integrity on real-time PCR results – tips to achieve a true RNA profiling suitable for real-time PCR studies
2) Improved methods for cDNA synthesis, optimized for real-time PCR
3) Real-time PCR analysis:
• Real-time PCR essentials and background information on different quantification strategies
• SYBR Green real-time PCR – factors influencing specificity
• Introduction to probe technology
• New, fast and efficient real-time PCR solutions
Nucleic Acid Quantification from FFPE Samples – Are You Doing it Right?QIAGEN
Formalin-fixation and paraffin-embedding is a standard method for long-term preservation of tissue biopsies and these stored samples are a valuable tool for studying diseases such as cancer, especially when they are histologically and pathologically well characterized, and follow-up clinical data is available. The quality of nucleic acids extracted from FFPE samples is influenced by a number of factors, including how the samples were handled before, during and after fixation and embedding. Moreover, there are several difficulties when purifying nucleic acids from FFPE samples as the chemicals and temperature used during the process can degrade the DNA.
In this webinar, we will discuss the variability in quantity and purity of DNA purified from FFPE material. We will show data from different quantification and quality control methods, demonstrate the impact of inaccurate quantification on downstream results and discuss how to overcome these challenges.
Webinar: Evaluating Viral Clearance for Continuous ProcessesMilliporeSigma
Participate in the interactive webinar now: http://bit.ly/ViralClearanceWebinar
Is viral clearance a hurdle to implementing continuous processing? We’ll share virus spiking alternatives that may pave the way for effectively evaluating viral clearance by chromatography steps in a continuous process.
Explore our webinar library: www.emdmillipore.com/webinars
Optimal RNAlater® incubation and removal conditions prior to isolation of tot...QIAGEN
RNA is highly sensitive to degradation. Handling methods and prolonged storage of cells can greatly affect the quality of the RNA that can be later isolated. Contamination with RNases is the most significant problem, especially as they are so ubiquitous in the environment. They can degrade RNA to the point where results of downstream analyses become meaningless.
Submerging cells in RNAlater, an RNA stabilization reagent, helps to stabilize the RNA within the cells and prevent degradation, supporting accurate downstream gene expression analyses. However, to avoid any interference from any RNAlater components in isolation and analyses, cells must be pelleted and the reagent must be removed. The separation of cells from excess RNAlater via centrifugation is impeded due to the higher density of the reagent compared to standard culture medium. This means it requires higher centrifugal forces, which might damage cells due to increased shearing forces, leading to reduced RNA yield. The aim of this study was to establish the optimal conditions for the recovery of cells from RNAlater after RNA stabilization for maximum RNA yield and integrity.
Process Development for Cell Therapy and Viral Gene TherapyMerck Life Sciences
Today’s viral vector manufacturing processes remain challenging. Process development is a critical enabler to bring safe, effective, sustainable products to market to address patient needs. When done properly, it can reduce the timeline of the project and the cost of producing the therapeutic product.
The webinar discusses our strategies for developing lentivirus and adeno associated virus (AAV) and the impact these early decisions can have on commercial readiness.
Watch the interactive webinar now: https://bit.ly/2VplwQq
PCR Array Data Analysis Tutorial: qPCR Technology Webinar Series Part 3QIAGEN
Using actual PCR Array data, this slidedeck presents an easy-to-use and free web-based data analysis tool to calculate fold-differences in gene expression from your raw real-time PCR threshold cycles. Learn how you can look at your results in different formats, including heat map, scatter, volcano, clustergram and multigroup plot.
Stable 16 year storage of DNA purified with the QIAamp® DNA Blood mini kit - ...QIAGEN
In this application note, we describe the success of the QIAamp DNA Blood Mini Kit in the preparation of highly stable DNA,as evidenced by 16-year storage data. We also report the best storage conditions for maximal protection against degradation.
Simultaneous Isolation of RNA & DNA from one FFPE SampleQIAGEN
Worldwide, there are millions of tissue samples archived in tissue biobanks and biorepositories. These samples are extremely valuable for pharmacological and biomedical research and companion diagnostics, due to the linkage to patient history. The vast majority of archived tissue samples are formalin-fixed and paraffin-embedded (FFPE), since formalin is the standard fixative for tissue samples.
FFPE blocks serve as an excellent source for histomorphology studies, but their use in molecular studies is challenging, due to crosslinking and fragmentation caused by fixation, processing, embedding, and storage conditions. For reliable comparison of genomic and transcriptomic data from heterogeneous samples and to spare sample material, purification of DNA and RNA from
the same sample is essential. This is particularly important when working with tumorous tissues, which contain a heterogeneous distribution of healthy and malignant cells.
Liquid biopsy: Overcome Challenges of Circulating DNA with Automated and Stan...QIAGEN
Circulating cell-free DNA (ccfDNA) originating from malignant tumors, a developing fetus and also from inflammatory tissues, is present in the cell-free nucleic acids in plasma, serum and other body fluids and is considered a “liquid biopsy”. Access to ccfDNA for analysis allows for specific detection of certain disease states based on a simple blood sample. Circulating cell-free DNA shows distinctive properties – it is present mostly as shorter fragments of less than 500 bp and the concentration of ccfDNA in a plasma or serum sample is low (approximately 1–100 ng/ml) compared to cellular materials and varies considerably between different individuals.
Because of their fragmented nature and low concentration, ccfDNA presents a particular challenge for efficient extraction / purification and quantification, such as by qPCR. We present data on solutions for the following critical problems concerning the purification of ccfDNA for research and molecular diagnostic applications:
• Pre-analytical workflow (blood processing) for analyzing ccfDNA
• Optimization of ccfDNA extraction from plasma samples: low target concentrations require efficient ccfDNA enrichment from larger sample volumes
• Novel automated extraction of ccfDNA using the QIAsymphony SP instrument for liquid biopsy diagnostic applications.
HIV Vaccines Process Development & Manufacturing - Pitfalls & PossibilitiesKBI Biopharma
Originally presented at the HIV Vaccine Manufacturing Workshop –July 19th& 20th, 2017 by Abhinav A. Shukla, Ph.D.Senior Vice PresidentDevelopment & ManufacturingKBI Biopharma, Durham NC
Real-Time PCR
The Polymerase Chain Reaction (PCR) is a process for the
amplification of specific fragments of DNA.
Real-Time PCR a specialized technique that allows a PCR reaction
to be visualized “in real time” as the reaction progresses.
Real-Time PCR allows us to measure minute amounts of DNA
sequences in a sample.
Uses of Real-Time PCR
Real-Time PCR has become a cornerstone of molecular biology:
Gene expression analysis
Cancer research
Drug research
Disease diagnosis and management
Viral quantification
Food testing
Testing of GMO food
Animal and plant breeding
Gene copy number
A targeted subgenomic approach for phylogenomics based on microfluidic pcr an...SimonUribeConvers
We describe a new method for generating large amounts of genomic data using microfluidic PCR and high-throughput sequencing. We also have a new way to process the raw information and are able to recover not only 50,000 base pairs for ~500 samples but also the alleles for each individual sample.
This is a presentation that I gave at the conference Botany2014 in Boise, Idaho, USA.
Pcr technology and its importance in covid 19 pandemicAnupam Maity
Since the discovery of the PCR technology, its application in the various fields is increased gradually. Based on to this principle, many variations of the PCR have been established. Year by year, it is upgraded very much. It is established as a most common and accurate technique for the detection of the various diseases in the field of medicine. Now it is a ‘Gold standard’ for the detection of covid-19 also, which is much needed to contain the spread of the virus. Though various detection techniques are there for detection, but real time RT-PCR (variation of PCR) is most reliable. Viral detection is based on a simple principle of nucleic acid (viral) amplification. Various manufacturing companies are manufacturing the PCR instrument. Though the accuracy of the instruments are slightly differ to each other.
Biosafety in Gene Therapy: Applying the latest regulatory guidance for RCL te...Merck Life Sciences
Watch the presentation of this webinar here: https://bit.ly/33WUiqE
Ensuring the safety and quality of your lentiviral vector is of the utmost importance. Attend this webinar to learn about testing strategies to monitor replication competent lentivirus. You will also hear about recent changes in regulatory guidance with regards to sample types and volumes tested.
The use of lentivirus vectors to produce groundbreaking gene therapies is on the rise. Ensuring the biosafety and quality of these vectors is achieved through a multi-tiered testing approach.
For lentivirus-based therapies, generation of replication competent particles is a potential risk. While improvements in design and manufacturing have decreased the probability of producing replication competent viruses, regulatory agencies provide guidelines to test for their presence at multiple stages in production. This webinar reviews the strategies for monitoring replication competent lentiviruses. We describe current methods and address: Sample types, testing volumes, and expected results.
In this webinar, you will learn about:
• The latest FDA regulatory guidelines on replication competent lentivirus (RCL) testing
• Methods used to monitor RCL
• Considerations on sample type and volume requirements
Biosafety in Gene Therapy: Applying the latest regulatory guidance for RCL te...MilliporeSigma
Watch the presentation of this webinar here: https://bit.ly/33WUiqE
Ensuring the safety and quality of your lentiviral vector is of the utmost importance. Attend this webinar to learn about testing strategies to monitor replication competent lentivirus. You will also hear about recent changes in regulatory guidance with regards to sample types and volumes tested.
The use of lentivirus vectors to produce groundbreaking gene therapies is on the rise. Ensuring the biosafety and quality of these vectors is achieved through a multi-tiered testing approach.
For lentivirus-based therapies, generation of replication competent particles is a potential risk. While improvements in design and manufacturing have decreased the probability of producing replication competent viruses, regulatory agencies provide guidelines to test for their presence at multiple stages in production. This webinar reviews the strategies for monitoring replication competent lentiviruses. We describe current methods and address: Sample types, testing volumes, and expected results.
In this webinar, you will learn about:
• The latest FDA regulatory guidelines on replication competent lentivirus (RCL) testing
• Methods used to monitor RCL
• Considerations on sample type and volume requirements
Understanding and controlling for sample and platform biases in NGS assaysCandy Smellie
What is the impact of assay failure in your laboratory and how do you monitor for it?
The advancement of next-generation sequencing has provided invaluable resources to researchers in multiple industries and disciplines, and will be a major driver during the personalized medicine revolution that is upon us. However, while the cost of generating sequencing data continues to decrease this does not take into account the significant costs associated with the infrastructure and expertise that are required to develop a robust, routine NGS pipeline.
Specifically, as predicted by Sboner, et al in 2011, the cost of the sequencing portion of the experiment continues to decrease and the costs associated with upfront experimental design and downstream analysis dominate the cost of each assay. This is true whether you are performing a pre-clinical R&D project, and perhaps even more so for clinical assays. In the paper, the authors note the unpredictable and considerable ‘human time’ spent on the upstream design and downstream analysis. Here at Horizon, we aim to develop tools that help researchers and clinicians optimize these workflows to make NGS more reliable and ultimately, more affordable by streamlining these resource intensive areas.
High Throughput Bioreactor Mimetic in Early and Late Stage Process DevelopmentKBI Biopharma
A presentation by KBI Scientist Shahid Rameez, Ph.D. at the American Chemical Society Annual Meeting– Biochemical Technology (BIOT) Division, New Orleans, LA
Microfluidic PCR Devices for DNA Amplification PresentationFarid MUSA
Presentation on the latest research on "Microfluidic PCR Devices for DNA Amplification". Might be helpful for students and others who are interested.
Report Included
Microfluidic PCR Devices for DNA Amplification ReportFarid MUSA
Report on the latest research on "Microfluidic PCR Devices for DNA Amplification". Might be helpful for students and others who are interested.
Report Included
Similar to Dario Lijtmaer - PCR Amplification (20)
How to Make a Field invisible in Odoo 17Celine George
It is possible to hide or invisible some fields in odoo. Commonly using “invisible” attribute in the field definition to invisible the fields. This slide will show how to make a field invisible in odoo 17.
Honest Reviews of Tim Han LMA Course Program.pptxtimhan337
Personal development courses are widely available today, with each one promising life-changing outcomes. Tim Han’s Life Mastery Achievers (LMA) Course has drawn a lot of interest. In addition to offering my frank assessment of Success Insider’s LMA Course, this piece examines the course’s effects via a variety of Tim Han LMA course reviews and Success Insider comments.
Operation “Blue Star” is the only event in the history of Independent India where the state went into war with its own people. Even after about 40 years it is not clear if it was culmination of states anger over people of the region, a political game of power or start of dictatorial chapter in the democratic setup.
The people of Punjab felt alienated from main stream due to denial of their just demands during a long democratic struggle since independence. As it happen all over the word, it led to militant struggle with great loss of lives of military, police and civilian personnel. Killing of Indira Gandhi and massacre of innocent Sikhs in Delhi and other India cities was also associated with this movement.
Acetabularia Information For Class 9 .docxvaibhavrinwa19
Acetabularia acetabulum is a single-celled green alga that in its vegetative state is morphologically differentiated into a basal rhizoid and an axially elongated stalk, which bears whorls of branching hairs. The single diploid nucleus resides in the rhizoid.
Synthetic Fiber Construction in lab .pptxPavel ( NSTU)
Synthetic fiber production is a fascinating and complex field that blends chemistry, engineering, and environmental science. By understanding these aspects, students can gain a comprehensive view of synthetic fiber production, its impact on society and the environment, and the potential for future innovations. Synthetic fibers play a crucial role in modern society, impacting various aspects of daily life, industry, and the environment. ynthetic fibers are integral to modern life, offering a range of benefits from cost-effectiveness and versatility to innovative applications and performance characteristics. While they pose environmental challenges, ongoing research and development aim to create more sustainable and eco-friendly alternatives. Understanding the importance of synthetic fibers helps in appreciating their role in the economy, industry, and daily life, while also emphasizing the need for sustainable practices and innovation.
A Strategic Approach: GenAI in EducationPeter Windle
Artificial Intelligence (AI) technologies such as Generative AI, Image Generators and Large Language Models have had a dramatic impact on teaching, learning and assessment over the past 18 months. The most immediate threat AI posed was to Academic Integrity with Higher Education Institutes (HEIs) focusing their efforts on combating the use of GenAI in assessment. Guidelines were developed for staff and students, policies put in place too. Innovative educators have forged paths in the use of Generative AI for teaching, learning and assessments leading to pockets of transformation springing up across HEIs, often with little or no top-down guidance, support or direction.
This Gasta posits a strategic approach to integrating AI into HEIs to prepare staff, students and the curriculum for an evolving world and workplace. We will highlight the advantages of working with these technologies beyond the realm of teaching, learning and assessment by considering prompt engineering skills, industry impact, curriculum changes, and the need for staff upskilling. In contrast, not engaging strategically with Generative AI poses risks, including falling behind peers, missed opportunities and failing to ensure our graduates remain employable. The rapid evolution of AI technologies necessitates a proactive and strategic approach if we are to remain relevant.
The Roman Empire A Historical Colossus.pdfkaushalkr1407
The Roman Empire, a vast and enduring power, stands as one of history's most remarkable civilizations, leaving an indelible imprint on the world. It emerged from the Roman Republic, transitioning into an imperial powerhouse under the leadership of Augustus Caesar in 27 BCE. This transformation marked the beginning of an era defined by unprecedented territorial expansion, architectural marvels, and profound cultural influence.
The empire's roots lie in the city of Rome, founded, according to legend, by Romulus in 753 BCE. Over centuries, Rome evolved from a small settlement to a formidable republic, characterized by a complex political system with elected officials and checks on power. However, internal strife, class conflicts, and military ambitions paved the way for the end of the Republic. Julius Caesar’s dictatorship and subsequent assassination in 44 BCE created a power vacuum, leading to a civil war. Octavian, later Augustus, emerged victorious, heralding the Roman Empire’s birth.
Under Augustus, the empire experienced the Pax Romana, a 200-year period of relative peace and stability. Augustus reformed the military, established efficient administrative systems, and initiated grand construction projects. The empire's borders expanded, encompassing territories from Britain to Egypt and from Spain to the Euphrates. Roman legions, renowned for their discipline and engineering prowess, secured and maintained these vast territories, building roads, fortifications, and cities that facilitated control and integration.
The Roman Empire’s society was hierarchical, with a rigid class system. At the top were the patricians, wealthy elites who held significant political power. Below them were the plebeians, free citizens with limited political influence, and the vast numbers of slaves who formed the backbone of the economy. The family unit was central, governed by the paterfamilias, the male head who held absolute authority.
Culturally, the Romans were eclectic, absorbing and adapting elements from the civilizations they encountered, particularly the Greeks. Roman art, literature, and philosophy reflected this synthesis, creating a rich cultural tapestry. Latin, the Roman language, became the lingua franca of the Western world, influencing numerous modern languages.
Roman architecture and engineering achievements were monumental. They perfected the arch, vault, and dome, constructing enduring structures like the Colosseum, Pantheon, and aqueducts. These engineering marvels not only showcased Roman ingenuity but also served practical purposes, from public entertainment to water supply.
Model Attribute Check Company Auto PropertyCeline George
In Odoo, the multi-company feature allows you to manage multiple companies within a single Odoo database instance. Each company can have its own configurations while still sharing common resources such as products, customers, and suppliers.
Read| The latest issue of The Challenger is here! We are thrilled to announce that our school paper has qualified for the NATIONAL SCHOOLS PRESS CONFERENCE (NSPC) 2024. Thank you for your unwavering support and trust. Dive into the stories that made us stand out!
Macroeconomics- Movie Location
This will be used as part of your Personal Professional Portfolio once graded.
Objective:
Prepare a presentation or a paper using research, basic comparative analysis, data organization and application of economic information. You will make an informed assessment of an economic climate outside of the United States to accomplish an entertainment industry objective.
1. Short course on DNA barcoding methods
November 29, 2011
PCR amplification
Darío Lijtmaer
Museo Argentino de Ciencias Naturales “Bernardino Rivadavia”
2. Organization of the talk
1) Equipment needed for PCR amplification.
2) Overview of PCR protocols.
3) Product verification: agarose gels.
4) Minimizing the risks of contamination.
5) Shipping and storing DNA extracts.
6) Discussion and questions.
3. Equipment: basic for a small-sized facility
- Hundreds or few thousands of barcodes produced per year.
- Tube scale.
Incubator Autoclave
Thermocycler
Pipettes Disposables and
reagents
Vortex
4. Equipment: medium-sized and high throughput facilities
- Many thousand barcodes produced per year.
- Plate scale.
Incubator Autoclave
Thermocycler
Pipettes Disposables and
reagents
Vortex
5. Overview of amplification protocols
None of the lab protocols/procedures are necessarily different from those
used for other mitochondrial markers or other projects.
6. Overview of amplification protocols
None of the lab protocols/procedures are necessarily different from those
used for other mitochondrial markers or other projects.
However...
a) Due to the scale of the project efforts are made to reduce the cost of
the molecular steps of the pipeline (e.g. small PCR volumes).
b) Certain requirements are needed to achieve the barcode data standard
(e.g. minimum length).
As a consequence innovations and development of new, more efficient
protocols/proceedures are frequent in the context of the project.
7. Overview of amplification protocols: CCDB
Animals: COI
This protocol can be used with:
• Individual tubes in small-sized
facilities.
• 96 well plates in medium-sized or
high throughput facilities.
www.barcodeoflife.org
8. Overview of amplification protocols: CCDB
Plants and fungi
This protocol can be used with:
• Individual tubes in small-sized
facilities.
• 96 well plates in medium-sized or
high throughput facilities.
www.barcodeoflife.org
9. Overview of amplification protocols: PCR mix (CCDB)
Small PCR volumes: 12.5 ml for most reactions (e.g. COI in animals and
rbcL in plants), 6.25 ml for matK (plants).
• Cost-efficient.
10. Overview of amplification protocols: PCR mix (CCDB)
Small PCR volumes: 12.5 ml for most reactions (e.g. COI in animals and
rbcL in plants), 6.25 ml for matK (plants).
• Cost-efficient.
Trehalose is used as part of the PCR mix.
• It allows freezing aliquots of the mix (useful for high throughput
facilities).
• It estabilizes the reaction.
11. Overview of amplification protocols: PCR mix (CCDB)
Small PCR volumes: 12.5 ml for most reactions (e.g. COI in animals and
rbcL in plants), 6.25 ml for matK (plants).
• Cost-efficient.
Trehalose is used as part of the PCR mix.
• It allows freezing aliquots of the mix (useful for high throughput
facilities).
• It estabilizes the reaction.
Platinum taq polymerase.
• High success and band intensity, less optimization needed.
• Hot start .
• Stable at room temperature.
12. Overview of amplification protocols: PCR mix (other tips)
BSA can be added to the PCR mix to improve PCR results. This is done at
Smithsonian LAB with invertebrate samples and in the African Centre for
DNA Barcoding with plant samples.
13. Overview of amplification protocols: PCR mix (other tips)
BSA can be added to the PCR mix to improve PCR results. This is done at
Smithsonian LAB with invertebrate samples and in the African Centre for
DNA Barcoding with plant samples.
DMSO can also be added to improve PCR results with difficult samples.
14. Overview of amplification protocols: primers
Primer choice is a key aspect of PCR success and probably the only aspect
of amplification that is taxon-dependent.
15. Overview of amplification protocols: primers
Primer choice is a key aspect of PCR success and probably the only aspect
of amplification that is taxon-dependent.
Ideal situation: universal primers.
Real world: various sets of primers are to be used (and sometimes
combined) depending on the taxonomic group. There is also more than
one option for each group.
16. Overview of amplification protocols: primers
Primer choice is a key aspect of PCR success and probably the only aspect
of amplification that is taxon-dependent.
Ideal situation: universal primers.
Real world: various sets of primers are to be used (and sometimes
combined) depending on the taxonomic group. There is also more than
one option for each group.
17. Overview of amplification protocols: primers
Primer choice is a key aspect of PCR success and probably the only aspect
of amplification that is taxon-dependent.
Ideal situation: universal primers.
Real world: various sets of primers are to be used (and sometimes
combined) depending on the taxonomic group. There is also more than
one option for each group.
We included as part of the complementary materials:
the list of primers that are used at the CCDB with each taxonomic
group, the sequence of those primers and the thermocycling program that
is used with each primer.
the list of primers and the thermocycling program used at the
Smithsonian LAB.
19. Overview of amplification protocols
PCR protocols also depend on the quality of the samples used.
For example, samples with potentially degraded DNA, such as relatively
old museum samples, require special primers designed to amplify
shorter, overlapping fragments (e.g. 200 bp long).
20. Overview of amplification protocols
PCR protocols also depend on the quality of the samples used.
For example, samples with potentially degraded DNA, such as relatively
old museum samples, require special primers designed to amplify
shorter, overlapping fragments (e.g. 200 bp long).
21. Product verification: agarose gels
PCR results are usually visualized in agarose gels.
Depending on the scale (and funding) home made gels or pre-cast gels are
used.
22. Product verification: agarose gels
PCR results are usually visualized in agarose gels.
Depending on the scale (and funding) home made gels or pre-cast gels are
used.
For plate-scales (medium scale or high-throughput) usually a threshold is
established (e.g. 75/95 bands at CCDB) and when a plate results are above
the threshold the entire plate is sequenced .
23. Mailing PCR products
If sequencing is not done on-site, PCR products are transferred to a plate
containing trehalose and dried before mailing them to a high throughput
facility.
24. Minimizing the risk of contamination
General practices
Clean workspace and sterile tips, tubes, etc.
25. Minimizing the risk of contamination
General practices
Clean workspace and sterile tips, tubes, etc.
Three sets of pipettes: one for extraction, one for preparing PCR and
one for PCR products (for example for gel loading).
26. Minimizing the risk of contamination
General practices
Clean workspace and sterile tips, tubes, etc.
Three sets of pipettes: one for extraction, one for preparing PCR and
one for PCR products (for example for gel loading).
If working with difficult samples, such as degraded DNA...
Special laboratory design (for example two separate doors that are
opened in sequence, presence of UV light).
Be extra-careful (for example, change gloves more often).