At the heart of every successful discovery lie the seeds of innovation. At QIAGEN, we are constantly developing new methods that allow researchers to gain forward momentum with their research. Whether you’re studying gene expression or performing viral RNA analysis, the success of your experiment depends on the ability to analyze your sample with the highest standards of sensitivity and specificity so that you can have confidence in your data. To help you generate valuable insights from gene expression profiling and viral RNA analysis, we introduce the brand new QIAGEN OneStep Ahead RT-PCR Kit – the first hot start reverse transcriptase kit on the market. Continuing the success story of its first-generation predecessor (QIAGEN OneStep RT-PCR Kit), the QIAGEN OneStep Ahead RT-PCR Kit is equipped with compelling new features that afford maximum convenience and ease of use, while delivering unmatched sensitivity and specificity. With a total reaction time of 1 hour, higher sequence accuracy and the ability to amplify amplicons of up to 4 kb without tedious optimization, you can get one step closer to publishing your findings with this new solution. For increased convenience, the kit comes in an all-in-one tube format along with a built-in pipetting control. Stay one step ahead of your peers and make significant advances in your research with the QIAGEN OneStep Ahead RT-PCR Kit! In this slidedeck, we introduce the new kit in detail and discuss its features and benefits.
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.
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
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
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.
Introduction to Real Time PCR (Q-PCR/qPCR/qrt-PCR): qPCR Technology Webinar S...QIAGEN
This slidedeck introduces the concepts of real-time PCR and how to conduct a real-time PCR assay. The topics that are covered include an overview of real-time PCR chemistries, protocols, quantification methods, real-time PCR applications and factors for success.
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.
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
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
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.
Introduction to Real Time PCR (Q-PCR/qPCR/qrt-PCR): qPCR Technology Webinar S...QIAGEN
This slidedeck introduces the concepts of real-time PCR and how to conduct a real-time PCR assay. The topics that are covered include an overview of real-time PCR chemistries, protocols, quantification methods, real-time PCR applications and factors for success.
The major agent of causes meningitis are :
1- 9802 gene fragment in Streptococcus pneumoniae.
2- omp P6 gene in Haemophilus influenzae .
3- ctrA gene in Neisseria meningitidis .
We can not detect these agents by Conventional lab. methods , and therefore we use PCR Techniques to ensure detect these agents
New Progress in Pyrosequencing for DNA MethylationQIAGEN
Pyrosequencing is a highly flexible technology that lets you rapidly analyze short- to medium-length sequences fast and quantitatively with high accuracy. The real-time, high-resolution sequence output makes the technology highly suitable for applications including complex mutation analysis, microbial identification and DNA methylation quantification.
The main bottleneck in Pyrosequencing has been limited sequence length, which is critical for some applications. Our new technology, software, and chemistry overcome this bottleneck and give sequence reads that are typically twice as long as those from previous PyroMark systems. The new PyroMark Q24 Advanced system also reduces background noise, improving quantification even at sites distant from the sequencing start. The new system is ideal for applications requiring analysis of longer sequences, such as DNA methylation analysis in epigenetic research, frequency determination in mutation analysis, and various de novo sequencing applications.
In this presentation, we will discuss the following applications and technology improvements:
• DNA methylation analysis at single base resolution at CpG and CpN sites
• Improved quantification of sequence variations at any sequence position
• Easy and improved base calling functionality
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
Introduction to real-Time Quantitative PCR (qPCR) - Download the slidesQIAGEN
This slidedeck introduces the concepts of real-time PCR and how to conduct a real-time PCR assay. The topics that are covered include an overview of real-time PCR chemistries, protocols, quantification methods, real-time PCR applications and factors for success.
The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2−ΔΔCT method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2−ΔΔCT method. In addition, we present the derivation and applications of two variations of the 2−ΔΔCT method that may be useful in the analysis of real-time, quantitative PCR data.
Critical Factors for Successful Real-Time PCR: Multiplex PCRQIAGEN
Multiplex end-point PCR is a powerful tool for genotyping and many other applications. QIAGEN’s multiplex PCR chemistry is optimized for reliable amplification of many different templates with high variability in copy numbers. Thus it enables very quick establishment of a new lab routine and instant success for your multiplex PCR strategy.
There is a set of critical factors which we recommend to be regarded for planning and performing this kind of PCR. These will be discussed in detail in the webinar. Additionally, our multiplex PCR chemistry has recently been gaining increasing popularity among scientists who are utilizing it for their next-generation sequencing workflows.
This slidedeck presents a simple and accurate real-time PCR system for relevant biological pathway- and disease-focused mRNA and long noncoding RNA (lncRNA) expression profiling. Learn about the stringent performance built into the technology to ensure its sensitivity, specificity, reproducibility and reliability. Application examples are also presented.
Learn about the power of LNA (Locked Nucleic Acid) technology and QIAGEN's LNA enhanced product portfolio for RNA and DNA research. Download the slide deck!
QIAGEN® Originals — Pure Plasmids, Genuine Kits - Learn moreQIAGEN
Plasmid DNA purification is one of the most commonly used methods in molecular biology. While being relatively simple to perform, purification of high-purity DNA is critical for reliable results in downstream applications.
As the innovator of plasmid DNA purification kits, QIAGEN has consistently set standards by providing faster preps, higher throughput, more convenience, and superior DNA quality for stringent applications. Learn more about the different plasmid purification solutions from QIAGEN for your research.
The major agent of causes meningitis are :
1- 9802 gene fragment in Streptococcus pneumoniae.
2- omp P6 gene in Haemophilus influenzae .
3- ctrA gene in Neisseria meningitidis .
We can not detect these agents by Conventional lab. methods , and therefore we use PCR Techniques to ensure detect these agents
New Progress in Pyrosequencing for DNA MethylationQIAGEN
Pyrosequencing is a highly flexible technology that lets you rapidly analyze short- to medium-length sequences fast and quantitatively with high accuracy. The real-time, high-resolution sequence output makes the technology highly suitable for applications including complex mutation analysis, microbial identification and DNA methylation quantification.
The main bottleneck in Pyrosequencing has been limited sequence length, which is critical for some applications. Our new technology, software, and chemistry overcome this bottleneck and give sequence reads that are typically twice as long as those from previous PyroMark systems. The new PyroMark Q24 Advanced system also reduces background noise, improving quantification even at sites distant from the sequencing start. The new system is ideal for applications requiring analysis of longer sequences, such as DNA methylation analysis in epigenetic research, frequency determination in mutation analysis, and various de novo sequencing applications.
In this presentation, we will discuss the following applications and technology improvements:
• DNA methylation analysis at single base resolution at CpG and CpN sites
• Improved quantification of sequence variations at any sequence position
• Easy and improved base calling functionality
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
Introduction to real-Time Quantitative PCR (qPCR) - Download the slidesQIAGEN
This slidedeck introduces the concepts of real-time PCR and how to conduct a real-time PCR assay. The topics that are covered include an overview of real-time PCR chemistries, protocols, quantification methods, real-time PCR applications and factors for success.
The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2−ΔΔCT method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2−ΔΔCT method. In addition, we present the derivation and applications of two variations of the 2−ΔΔCT method that may be useful in the analysis of real-time, quantitative PCR data.
Critical Factors for Successful Real-Time PCR: Multiplex PCRQIAGEN
Multiplex end-point PCR is a powerful tool for genotyping and many other applications. QIAGEN’s multiplex PCR chemistry is optimized for reliable amplification of many different templates with high variability in copy numbers. Thus it enables very quick establishment of a new lab routine and instant success for your multiplex PCR strategy.
There is a set of critical factors which we recommend to be regarded for planning and performing this kind of PCR. These will be discussed in detail in the webinar. Additionally, our multiplex PCR chemistry has recently been gaining increasing popularity among scientists who are utilizing it for their next-generation sequencing workflows.
This slidedeck presents a simple and accurate real-time PCR system for relevant biological pathway- and disease-focused mRNA and long noncoding RNA (lncRNA) expression profiling. Learn about the stringent performance built into the technology to ensure its sensitivity, specificity, reproducibility and reliability. Application examples are also presented.
Learn about the power of LNA (Locked Nucleic Acid) technology and QIAGEN's LNA enhanced product portfolio for RNA and DNA research. Download the slide deck!
QIAGEN® Originals — Pure Plasmids, Genuine Kits - Learn moreQIAGEN
Plasmid DNA purification is one of the most commonly used methods in molecular biology. While being relatively simple to perform, purification of high-purity DNA is critical for reliable results in downstream applications.
As the innovator of plasmid DNA purification kits, QIAGEN has consistently set standards by providing faster preps, higher throughput, more convenience, and superior DNA quality for stringent applications. Learn more about the different plasmid purification solutions from QIAGEN for your research.
Quality Control of RNA Samples - For Gene-Expression Results you Can Rely onQIAGEN
By their very nature RNA molecules, especially mRNA and regulator RNA, are labile and can be highly unstable and sensitive to heat, UV and RNase contamination. The quality, relevance and scientific impact of gene expression results directly depends on the ability to extract RNA without losing any fraction of interest, while preserving the integrity of the biological information it carries. RNA quality control is thus critical to ensure high-quality results and for turning these results into actionable insights with confidence.
In this webinar, we will introduce you to the main parameters influencing RNA-based assays and their respective impact on downstream applications, discuss how to monitor them and cover the advantages of automation for quality control along complex workflows.
Functional Analysis of miRNA: miRNA and its Role in Human Disease Webinar Ser...QIAGEN
This slideshow highlights the use of miRNA mimics, inhibitors and target protectors to increase, decrease and adjust the cellular concentration of miRNA and disrupt specific miRNA–mRNA interactions. A ready-to-use screening tool for identifying miRNA targets and info on how to predict mRNA targets using miRNA expression data are also highlighted.
Total RNA Discovery for RNA Biomarker Development WebinarQIAGEN
Precision medicine offers to transform patient care by targeting treatment to those with most to gain. To date the most significant advances have been at the level of DNA, for example, the use of somatic DNA alterations as diagnostic indicators of disease and for prediction of pharmacodynamic response. Development of RNA expression signatures as biomarkers has been more problematic. While RNA expression analysis has yielded valuable insights into the biological mechanisms of disease, RNA is a more unstable molecule than DNA, and more easily damaged or degraded during sample collection and isolation. In addition, RNA levels are inherently dynamic and gene expression signatures are extraordinarily complex. Recently, much progress has been made in identifying key changes in gene expression in cancer and other diseases, as well as identifying expression signatures in circulating nucleic acid that have the potential to be developed into diagnostic and prognostic indicators.
Meeting the challenges of miRNA research: miRNA and its Role in Human Disease...QIAGEN
miRNA plays a critical role in many biological processes such as differentiation and development, cell signaling, response to infection and more. This slideshow will cover the biology of miRNA, the key challenges associated with miRNA research and the latest advances in miRNA research technology.
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.
Noncoding RNAs in Cardiovascular Disease – Potential as Biomarkers and MoreQIAGEN
Cardiovascular diseases (CVD) are the leading cause of death worldwide, and are therefore the subject of intense, urgent research. Biomarkers could help physicians diagnose heart diseases early, for example, and better therapies could improve survival or healing following events like myocardial infarction. Small noncoding RNAs called microRNAs have recently stepped into the spotlight as circulating biomarkers for a number of diseases, and may also have utility in someday treating CVD more effectively. In this slide deck, we discuss why and how microRNAs are being investigated as biomarkers for CVD, as well as examining some recent findings in the field. Check it out to find out how scientists are investigating noncoding RNA involvement in CVD and how you can do the same in your laboratory!
Advanced miRNA Expression Analysis: miRNA and its Role in Human Disease Webin...QIAGEN
miRNAs are small functional RNAs, which regulate gene expression post-transcriptionally. The miScript miRNA PCR Array System is a sensitive and reliable technology for detection of mature miRNAs in any laboratory. In this slideshow, the challenges of miRNA data analysis and solutions that the miScript miRNA PCR Arrays provide for researchers interested in identifying miRNA from cells, tissues and FFPE samples are described. You will also learn how to use our GeneGlobe Data Analysis Center to identify miRNAs that may be important in your favorite biological pathway or disease.
Biofluid miRNA profiling: from sample to biomarker: miRNA and its Role in Hum...QIAGEN
Circulating miRNAs have great potential as biomarkers due to their aberrant expression in cancer and other diseases. However, miRNAs from body fluids are hard to obtain in amounts sufficient for detailed miRNome profiling. This slideshow describes an integrated, PCR-based system that reduces the amount of sample required for full miRNome profiling by several orders of magnitude and provides unparalleled reproducibility and precision. Detailed protocols are highlighted regarding RNA isolation, real-time quantification and data analysis for the assessment of serum, plasma, urine and cerebrospinal fluid samples. This system enables accurate miRNA analysis on the smallest of samples and opens up new possibilities for biomarker development.
Accelerate Your Discovery with QIAGEN Service Solutions for Biomarker Researc...QIAGEN
This slidedeck will highlight QIAGEN’s service capabilities in sample isolation, microarray and NGS-sequencing, qPCR panel and custom assay development and bioinformatics as we look at the identification of potential biomarkers and gene signatures. The applications of QIAGEN Service Core in microRNA discovery for toxicology markers in serum and plasma and in identification of RNA signatures for tumor stratification are featured. Learn how you can accelerate your research with QIAGEN service solutions.
The importance of controls and novel solutions for successful real-time qPCRQIAGEN
The increasing demand for streamlined, monitored and ultrafast qPCR procedures requires high-performance, real-time quantitative RT and PCR chemistries. Particularly, procedures utilizing generic kits for gene expression analysis should include in-process safety measures to avoid variables and control accuracy of procedures and results. This slidedeck presents innovative solutions for one-step and two-step RT-PCR that significantly enhance performance and reliability in qRT-PCR. The new QuantiNova kit family offers a combination of various integrated safety features to remove variables and prevent artifacts. Internal control RNA, removal of genomic DNA, room temperature set-up capability for RT-PCR and a built-in visual pipetting control verify accurate procedures, ensuring reliable gene expression profiling.
This slidedeck explains the principles of the technologies and shows data demonstrating performance in qRT-PCR. Find out how you can verify accurate performance in qRT-PCR and improve your results!
Summary table that compares the performance specifications of Illumina's new sequencers - HiSeq X and NextSeq with MiSeq and HiSeq 2500. Includes number of samples that can run on the different sequencers.
Guide to Molecular Cloning - Download the GuideQIAGEN
Molecular cloning can be sometimes tricky with significant challenges involved. Overcome the challenges with the essential knowledge and tips for successful cloning.
PCR - From Setup to Cleanup: A Beginner`s Guide with Useful Tips and Tricks -...QIAGEN
This End-Point PCR Beginner´s Guide will not only give you a comprehensive overview of tools and techniques to help you to get the most out of your samples, but also give you information on dedicated solutions and complete workflows on multiplex PCR and PCR fragment analysis.
Polymerase chain reaction (PCR) is a technique used in molecular biology to amplify a single copy or a few copies of a segment of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. It is an easy, cheap, and reliable way to repeatedly replicate a focused segment of DNA, a concept which is applicable to numerous fields in modern biology and related sciences.
Presentation on nested pcr . contain types of pcr, protocol of nested pcr, advantages of nested pcr, disadvantages of nested pcr, application of nested pcr ,pictorial representation of pcr.
It is a molecular biological technique.we can monitor the amplification of DNA or RNA sequence. we can aklso test Corona like disease trough this machine.
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.
Using methylation patterns to determine origin of biological material and ageQIAGEN
In this QIAGEN sponsored webinar, our guest speakers from the San Francisco Police Department (SFPD) Crime Lab and Florida International University (FIU) discuss their research on the potential of epigenetic methylation as a procedure for body fluid identification and age estimation from DNA left at crime scenes. Several approaches have been studied, including an analysis of methyl array data and an initial validation of procedures such as pyrosequencing and real-time PCR. The presentation focuses on a number of tissue-specific epigenetic markers for body fluid and age determination with a promise of future integration of these markers into the forensic lab due to the simplicity of analysis and the ease of application.
Learn more about the Pyrosequencing technology and our solutions at
https://www.qiagen.com/resources/technologies/pyrosequencing-resource-center/
Take lung cancer research to a new molecular dimensionQIAGEN
Circulating Tumor Cells (CTCs) can provide researchers with important new discoveries on the mechanism of cancer. Find out more about the latest technology that provides researchers the necessary tools to conduct CTC research in lung cancer.
Circulating Tumor Cells (CTCs) can provide researchers with important new discoveries on the mechanism of cancer. Find out more about the latest technology that provides researchers the necessary tools to conduct CTC research in AR-V7 related prostate cancer.
Take your RNA research to the next level with QIAGEN LNA tools!QIAGEN
Download the flyer!
Experience truly exceptional RNA research with QIAGEN's next-generation, LNA®-enhanced tools. LNA (Locked Nucleic Acid) oligos bind with much higher affinity and specificity to RNA targets than standard DNA and RNA oligos – This enables specific and sensitive detection of small RNAs and discrimination between highly similar
sequences.
An Approach to De-convolution of Mixtures in Touch DNA Samples. Download now!QIAGEN
7th QIAGEN Investigator Forum - Lisbon, March 8, 2018 . An Approach to De-convolution of Mixtures in Touch DNA Samples. Presenter: Lisa Dierig, Institute of Legal Medicine, Ulm
Assessment of Y chromosome degradation level using the Investigator® Quantipl...QIAGEN
Assessment of Y chromosome degradation level using the Investigator® Quantiplex® Pro RGQ Kit, presented by Dr. Tomasz Kupiec, Head of the Forensic Genetics Section, Institute of Forensic Research, Krakow, Poland on June 14, 2018.
ICMP MPS SNP Panel for Missing Persons - Michelle Peck et al.QIAGEN
Optimization and Performance of a Very Large MGS SNP Panel for Missing Persons, by Michelle Peck et al., International Commission on Mission Persons. Presented May 3, 2018, at the QIAGEN Investigator Forum, San Antonio, TX.
Exploring the Temperate Leaf Microbiome: From Natural Forests to Controlled E...QIAGEN
The aerial surfaces of plants, the phyllosphere, harbors a diverse community of microorganisms. The increasing awareness of the potential roles of phyllosphere microbial communities calls for a greater understanding of their structure and dynamics in natural and urban ecosystems. To do so, we characterized the community structure and assembly dynamics of leaf bacterial communities in natural temperate forests of Quebec by comparing the relative influence of host species identity, site, and time on phyllosphere bacterial community structure. Second, we tested the value of characterizing a tree’s complete phyllosphere microbial community through a single sample by measuring the intra-individual, inter-individual and interspecific variation in leaf bacterial communities. Third, we quantified the relationships among phyllosphere bacterial diversity, plant species richness, plant functional diversity and identity, and plant community productivity in a biodiversity-ecosystem function experiment with trees. Finally, we compared tree leaf bacterial communities in natural and urban environments, as well as along a gradient of increasing anthropogenic pressures. The work presented here thus offers an original assessment of the dynamics at play in the tree phyllosphere.
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.
The Microbiome of Research Animals : Implications for Reproducibility, Transl...QIAGEN
The human gut microbiota (GM) has emerged as a key factor in susceptibility to, as well as a potential biomarker of, several diseases and conditions. Similarly, researchers now appreciate that the GM of laboratory animals could affect the reproducibility and translatability of many disease models, including a complete loss of phenotype. While associations between characteristics of the GM and differential disease model phenotypes are of concern, they can also be viewed as sources of discovery related to disease pathogenesis. As such, there is considerable interest in factors that inadvertently influence the composition of the GM and methods of manipulating the GM prospectively to investigate such associations and standardize or optimize disease models. The webinar will present data on variables capable of influencing the GM of laboratory rodents citing several examples and animal models, considerations related to manipulation of the GM in mice and rats, and recent data supporting the use of “dirty” mice in biomedical research.
Building a large-scale missing persons ID SNP panel - Download the studyQIAGEN
In this webinar, we will take a look at a large-scale SNP-based forensic identification panel for DNA analysis with massively parallel sequencing (MPS). The panel was specifically designed for the challenges of identifying missing persons; where DNA is frequently highly degraded, and relationship tests may involve reference samples from across several generations and in a deficient pedigree.
Rapid DNA isolation from diverse plant material for use in Next Generation Se...QIAGEN
Isolation of DNA from plant material is often a tedious process which involves significant hands on time and leads to varying results due to the diverse nature of the material. Different parts of the plants as well as the plants themselves differ in both consistency of material and presence of inhibitory substances, making dependable isolation of DNA difficult.
Here, we developed a method for the efficient extraction of DNA from different plant types, including strawberry leaf, pine needle, grape leaf, and cotton and coffee seeds (workflow at right). A novel bead beating method and lysis chemistry led to more efficient sample lysis with minimal hands-on time and significantly increased DNA yield compared to conventional methods. Through the use of multiple technologies to improve removal of secondary metabolites, such as polyphenols, complex polysaccharides, alkaloids and tannins that may inhibit downstream applications, the isolated DNA was of high quality and purity.
The resulting DNA is suitable for immediate use in downstream reactions, including PCR, qPCR and Next Generation Sequencing based applications. Using this method we were further able to design a workflow that included DNA isolation, library preparation and bioinformatics analyses for the efficient detection of plant pathogens isolated from infected samples. With this, our protocol is a substantial improvement within workflows used for plant microbiome and plant pathology studies as well as in plant breeding and engineering.
Rapid extraction of high yield, high quality DNA from tissue samples - Downlo...QIAGEN
Genetic and genomic analysis from tissue samples requires the extraction of high quality DNA. Mechanical disruption methods such as bead milling provide high yield from tissue samples, but cause damage to the nucleic acids. Purely enzymatic methods such as proteinase K digestion can extract nucleic acid without damage, but require long incubation times, often proceeding overnight, and without approaching the yields achieved by mechanical disruption techniques. Thus a method is needed which can provide a rapid extraction of high yield, high quality DNA from tissue samples. See the new method.
Overcome the challenges of Nucleic acid isolation from PCR inhibitor-rich mic...QIAGEN
This presentation will focus on nucleic acid extraction tools developed by QIAGEN that facilitate accurate non-biased community analysis and eliminate common amplification problems via the depletion of endogenous polymerase inhibitors using our patented Inhibitor Removal Technology.
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.
Automated Nucleic Acid Purification from Diverse Sample types using dedicated...QIAGEN
This webinar will focus on the automation of QIAGEN’s new line of DNA and RNA sample prep kits for the microbiome. We will show how automation on the QIAcube enables efficient and reliable use of these samples for sensitive downstream applications such as qPCR and NGS. In addition, you will learn how to successfully use the CLC Microbial Genomics Module for metagenome sequencing and identification of microbial composition and diversity.
Dna Methylation Analysis in a Single Day - Download the SlidesQIAGEN
This webinar introduces the new PyroMark Q48 Autoprep system. Combined with the latest EpiTect Fast bisulfite conversion technology, the new PyroMark Q48 Autoprep can now provide highly automated methylation analysis in a single day.
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.
DNA Analysis - Basic Research : A Case StudyQIAGEN
Nucleic acid gel electrophoresis is a broadly used technique in all fields of basic life science research. The flexibility and versatility of the QIAxcel allows researchers to streamline and accelerate their molecular biology experiments. The sensitivity and resolution of capillary electrophoresis offers an excellent alternative to long or complex slab gel setup. A wide range of applications in basic research involving microsatellite analysis, mapping mutant genes, linkage analysis, and genotyping transgenes by PCR are all powerful molecular approaches for screening organisms and their genetic profiles. The QIAxcel Advanced provides precise and reliable results to accelerate these analyses and the research projects they are part of.
Characterizing the Microbiome of Neonates and Infants to explore associations...QIAGEN
This webinar slidedeck will focus on the acquisition and development of the preterm gut microbiome from birth and following discharge from intensive care. Specifically, the discussion will be around the association of the gut microbiome with necrotizing enterocolitis (NEC) and late onset sepsis (LOS), as well as the impact of birth mode. The other discussion points will be the analysis of multi-omic datasets, including the analysis of the airway microbiome and metabolome in infants hospitalized with bronchiolitis.
The Importance of Community Nursing Care.pdfAD Healthcare
NDIS and Community 24/7 Nursing Care is a specific type of support that may be provided under the NDIS for individuals with complex medical needs who require ongoing nursing care in a community setting, such as their home or a supported accommodation facility.
The dimensions of healthcare quality refer to various attributes or aspects that define the standard of healthcare services. These dimensions are used to evaluate, measure, and improve the quality of care provided to patients. A comprehensive understanding of these dimensions ensures that healthcare systems can address various aspects of patient care effectively and holistically. Dimensions of Healthcare Quality and Performance of care include the following; Appropriateness, Availability, Competence, Continuity, Effectiveness, Efficiency, Efficacy, Prevention, Respect and Care, Safety as well as Timeliness.
Antibiotic Stewardship by Anushri Srivastava.pptxAnushriSrivastav
Stewardship is the act of taking good care of something.
Antimicrobial stewardship is a coordinated program that promotes the appropriate use of antimicrobials (including antibiotics), improves patient outcomes, reduces microbial resistance, and decreases the spread of infections caused by multidrug-resistant organisms.
WHO launched the Global Antimicrobial Resistance and Use Surveillance System (GLASS) in 2015 to fill knowledge gaps and inform strategies at all levels.
ACCORDING TO apic.org,
Antimicrobial stewardship is a coordinated program that promotes the appropriate use of antimicrobials (including antibiotics), improves patient outcomes, reduces microbial resistance, and decreases the spread of infections caused by multidrug-resistant organisms.
ACCORDING TO pewtrusts.org,
Antibiotic stewardship refers to efforts in doctors’ offices, hospitals, long term care facilities, and other health care settings to ensure that antibiotics are used only when necessary and appropriate
According to WHO,
Antimicrobial stewardship is a systematic approach to educate and support health care professionals to follow evidence-based guidelines for prescribing and administering antimicrobials
In 1996, John McGowan and Dale Gerding first applied the term antimicrobial stewardship, where they suggested a causal association between antimicrobial agent use and resistance. They also focused on the urgency of large-scale controlled trials of antimicrobial-use regulation employing sophisticated epidemiologic methods, molecular typing, and precise resistance mechanism analysis.
Antimicrobial Stewardship(AMS) refers to the optimal selection, dosing, and duration of antimicrobial treatment resulting in the best clinical outcome with minimal side effects to the patients and minimal impact on subsequent resistance.
According to the 2019 report, in the US, more than 2.8 million antibiotic-resistant infections occur each year, and more than 35000 people die. In addition to this, it also mentioned that 223,900 cases of Clostridoides difficile occurred in 2017, of which 12800 people died. The report did not include viruses or parasites
VISION
Being proactive
Supporting optimal animal and human health
Exploring ways to reduce overall use of antimicrobials
Using the drugs that prevent and treat disease by killing microscopic organisms in a responsible way
GOAL
to prevent the generation and spread of antimicrobial resistance (AMR). Doing so will preserve the effectiveness of these drugs in animals and humans for years to come.
being to preserve human and animal health and the effectiveness of antimicrobial medications.
to implement a multidisciplinary approach in assembling a stewardship team to include an infectious disease physician, a clinical pharmacist with infectious diseases training, infection preventionist, and a close collaboration with the staff in the clinical microbiology laboratory
to prevent antimicrobial overuse, misuse and abuse.
to minimize the developme
CRISPR-Cas9, a revolutionary gene-editing tool, holds immense potential to reshape medicine, agriculture, and our understanding of life. But like any powerful tool, it comes with ethical considerations.
Unveiling CRISPR: This naturally occurring bacterial defense system (crRNA & Cas9 protein) fights viruses. Scientists repurposed it for precise gene editing (correction, deletion, insertion) by targeting specific DNA sequences.
The Promise: CRISPR offers exciting possibilities:
Gene Therapy: Correcting genetic diseases like cystic fibrosis.
Agriculture: Engineering crops resistant to pests and harsh environments.
Research: Studying gene function to unlock new knowledge.
The Peril: Ethical concerns demand attention:
Off-target Effects: Unintended DNA edits can have unforeseen consequences.
Eugenics: Misusing CRISPR for designer babies raises social and ethical questions.
Equity: High costs could limit access to this potentially life-saving technology.
The Path Forward: Responsible development is crucial:
International Collaboration: Clear guidelines are needed for research and human trials.
Public Education: Open discussions ensure informed decisions about CRISPR.
Prioritize Safety and Ethics: Safety and ethical principles must be paramount.
CRISPR offers a powerful tool for a better future, but responsible development and addressing ethical concerns are essential. By prioritizing safety, fostering open dialogue, and ensuring equitable access, we can harness CRISPR's power for the benefit of all. (2998 characters)
India Clinical Trials Market: Industry Size and Growth Trends [2030] Analyzed...Kumar Satyam
According to TechSci Research report, "India Clinical Trials Market- By Region, Competition, Forecast & Opportunities, 2030F," the India Clinical Trials Market was valued at USD 2.05 billion in 2024 and is projected to grow at a compound annual growth rate (CAGR) of 8.64% through 2030. The market is driven by a variety of factors, making India an attractive destination for pharmaceutical companies and researchers. India's vast and diverse patient population, cost-effective operational environment, and a large pool of skilled medical professionals contribute significantly to the market's growth. Additionally, increasing government support in streamlining regulations and the growing prevalence of lifestyle diseases further propel the clinical trials market.
Growing Prevalence of Lifestyle Diseases
The rising incidence of lifestyle diseases such as diabetes, cardiovascular diseases, and cancer is a major trend driving the clinical trials market in India. These conditions necessitate the development and testing of new treatment methods, creating a robust demand for clinical trials. The increasing burden of these diseases highlights the need for innovative therapies and underscores the importance of India as a key player in global clinical research.
Medical Technology Tackles New Health Care Demand - Research Report - March 2...pchutichetpong
M Capital Group (“MCG”) predicts that with, against, despite, and even without the global pandemic, the medical technology (MedTech) industry shows signs of continuous healthy growth, driven by smaller, faster, and cheaper devices, growing demand for home-based applications, technological innovation, strategic acquisitions, investments, and SPAC listings. MCG predicts that this should reflects itself in annual growth of over 6%, well beyond 2028.
According to Chris Mouchabhani, Managing Partner at M Capital Group, “Despite all economic scenarios that one may consider, beyond overall economic shocks, medical technology should remain one of the most promising and robust sectors over the short to medium term and well beyond 2028.”
There is a movement towards home-based care for the elderly, next generation scanning and MRI devices, wearable technology, artificial intelligence incorporation, and online connectivity. Experts also see a focus on predictive, preventive, personalized, participatory, and precision medicine, with rising levels of integration of home care and technological innovation.
The average cost of treatment has been rising across the board, creating additional financial burdens to governments, healthcare providers and insurance companies. According to MCG, cost-per-inpatient-stay in the United States alone rose on average annually by over 13% between 2014 to 2021, leading MedTech to focus research efforts on optimized medical equipment at lower price points, whilst emphasizing portability and ease of use. Namely, 46% of the 1,008 medical technology companies in the 2021 MedTech Innovator (“MTI”) database are focusing on prevention, wellness, detection, or diagnosis, signaling a clear push for preventive care to also tackle costs.
In addition, there has also been a lasting impact on consumer and medical demand for home care, supported by the pandemic. Lockdowns, closure of care facilities, and healthcare systems subjected to capacity pressure, accelerated demand away from traditional inpatient care. Now, outpatient care solutions are driving industry production, with nearly 70% of recent diagnostics start-up companies producing products in areas such as ambulatory clinics, at-home care, and self-administered diagnostics.
Medical Technology Tackles New Health Care Demand - Research Report - March 2...
One Step Ahead for Your RT-PCR
1. Sample to Insight
One Step Ahead for Your RT-PCR
Dr. Bernd Willems, Global Product Manager
1
2. Sample to Insight
Legal disclaimer
One Step Ahead for Your RT-PCR 2
• QIAGEN products shown here are intended for molecular biology
applications. These products are not intended for the diagnosis,
prevention or treatment of a disease.
• For up-to-date licensing information and product-specific
disclaimers, see the respective QIAGEN kit handbook or user
manual. QIAGEN kit handbooks and user manuals are available
at www.QIAGEN.com or can be requested from QIAGEN
Technical Services or your local distributor.
6. Sample to Insight
Introduction
6
The advantages of one-step RT-PCR
• Streamlined workflow
• Save time / effort / money
• One-tube format reduces risk of contamination
• More robust and reliable results
One Step Ahead for Your RT-PCR
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Eight pitfalls and one solution
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1. Influence of annealing temperature on specificity
2. Nonspecific amplification during reaction setup
3. Risk of RNase contamination
4. PCR-borne mutations
5. RNA secondary structure and high GC-content
6. Pipetting errors
7. False negative results
8. Target RNAconcentration threshold
Eight pitfalls for all who are performing one-stepRT-PCR
One Step Ahead for Your RT-PCR
9. Sample to Insight
Eight pitfalls and one solution
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Pitfall one:Influence of annealing temperature on specificity
The QIAGEN OneStep Ahead RT-PCRkit’s solution:
K+ and NH4+ -unique dual cation formulation
One Step Ahead for Your RT-PCR
10. Sample to Insight
Eight pitfalls and one solution
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Key technologies to improve annealing specificity: The buffer
K+ and NH4+ -unique dual cation formulation:
Stabilization by potassium cations
interacting with the phosphate backbone
Destabilization by ammonium cations
interacting with the hydrogen bonds of
nucleotide mismatches
Pitfall one:Influence of annealing temperature on specificity (cont’d)
One Step Ahead for Your RT-PCR
11. Sample to Insight
Eight pitfalls and one solution
11
Key technologies to improve annealing specificity: The buffer
K+ and NH4
+ -unique dual cation formulation:
Thus, only specific binding remains
stable…
…which guarantees the highest
degree of specificity
Pitfall one:Influence of annealing temperature on specificity (cont’d)
One Step Ahead for Your RT-PCR
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Eight pitfalls and one solution
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Indicated amounts of HeLa total RNA (in ng) w ere used as template for amplification of
EIF2B4 (107 bp) in duplicate, according to the suppliers’ instructions. Green arrow s
indicate specific product, red arrow s indicate primer‐dimers, purple arrow s indicate
nonspecific amplification products. Analysis w as performed using the QIAxcel.
Pitfall one:Influence of annealing temperature on specificity (cont’d)
Comparing the QIAGEN Kit performance with competitors
One Step Ahead for Your RT-PCR
13. Sample to Insight
Eight pitfalls and one solution
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One annealing temperature for all
assays.
45°C 60°C
Many assays work over an
annealing temperature range from
45-60°C.
-55°C proofed to be a universally
recommendable annealing
temperature.
Pitfall one:Influence of annealing temperature on specificity (cont’d)
One Step Ahead for Your RT-PCR
14. Sample to Insight
Eight pitfalls and one solution
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Pitfall two:Nonspecific amplification during reaction setup
• Residual activity of RT enzyme at room temperature leads to unspecific amplification at primer dimers
• These are competing for resources with the target of interest
The QIAGEN OneStep Ahead RT-PCRkit’s solution:
• The unique RT-Blocker keeps the RT-enzymes completely inactive at ambient temperatures
• It only dissociates to activate the RT-enzyme when temperature reaches the catalytic optimum for
reverse transcription
• This enables convenient room temperature setup and even allows keeping the reactions at room
temperature for up to 2h before cycling
• This enables use of the kit in automated robotic workflows
One Step Ahead for Your RT-PCR
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Eight pitfalls and one solution
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Pitfall two:Nonspecific amplification during reaction setup (cont’d)
One Step Ahead for Your RT-PCR
16. Sample to Insight
Eight pitfalls and one solution
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Pitfall three:Risk of RNAse contamination
• RNAs are highly fragile molecules; RNA cleaving RNases are widely dispersed in
our environment
• Rnases can easily contaminate your sample material and destroy precious RNA
The QIAGEN OneStep Ahead RT-PCRkit’s solution:
• Included RNase inhibitor prevents RNA decay caused by accidental RNase
contamination
• One-step RT-PCR format ensures minimal contamination due with a one-tube
approach
One Step Ahead for Your RT-PCR
17. Sample to Insight
Eight pitfalls and one solution
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Pitfall four:PCR-borne mutations
• PCR amplification using normal Taq DNA polymerase is prone to error in the DNA replication
process
• Typical rate of point mutations caused by Taq is 1 in 9,000
The QIAGEN OneStep Ahead RT-PCR kit’s solution:
• An additional high-fidelity enzyme with a 3' to 5' exonuclease proofreading activity elevates
the overall fidelity of the PCR step
• Furthermore it improves the processivity to allow for amplification of longer targets up to 4kb
HeLa total RNA (100 and 10
ng) w as used as a template for
amplification of RANBP2 (3
kbp).
One Step Ahead for Your RT-PCR
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Eight pitfalls and one solution
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Pitfall five:RNAsecondarystructure and high GC-content
QIAGEN Supplier T
• RNA with complex secondary structure can cause the reverse transcriptase to stop or dissociate from the
RNA template
• Furthermore, the reverse transcriptasecan skip over looped-out regions of RNA
• GC-rich sequences cause tight association of RNA:DNA hybrids which interfere with primer binding and
thus prevent amplification
The QIAGEN OneStep Ahead RT-PCRkit’s solution:
• The combination of Omniscript and Sensiscript ensure highest affinity to any RNA template
• Q-Solution facilitates reverse transcription and amplification of templates with a high GC-content or a
high degree of secondary structure
• Often enables or improves a suboptimal PCR caused by templates that have a high degree of
secondary structure or that have a GC-rich sequence
• Used at just one working concentration,which has been optimized for the requirements of multiplex
PCR
Figure 3: HeLa total RNA (100 ng) was
used as template for amplification of
TNFRI (581 bp) with a GC-ratio of
67.1%. Reactions were performed in
triplicate, according to the suppliers’
instructions. Green arrows indicate
specific product. Q-Solution was used
for the QIAGEN reactions.
One Step Ahead for Your RT-PCR
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Eight pitfalls and one solution
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Pitfall six: Pipetting errors
• A simple yet very effective solution: Optional tracking
system for visual indication of correct pipetting
• Consists of two inert dyes, a yellow Master Mix Tracer
and a blue Template Tracer which can be added to
the master mix and the template, respectively
• When the template is pipetted into the master mix, the
solution turns from yellow to green
• Both dyes also serve as gel tracking dyes during
electrophoresis.
• Pipetting errors are a common problem in daily lab routine
• When pipetting colorless solutions on a large scale (e.g. 96 well plates), keeping track is difficult
The QIAGEN OneStep Ahead RT-PCR kit’s solution:
One Step Ahead for Your RT-PCR
20. Sample to Insight
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Color switch after template addition
1 1 2 3 4
1: no template added
2: 14 µl template added
3: 5 µl template added
4: 2 µl template added
1 1 2 3 4
10 µl of 25 µl reaction loaded
1,5% agarose gel
~ 50 bp
~ 4 kbp
1 2 3 4
Eight pitfalls and one solution
Template Master Mix
One Step Ahead for Your RT-PCR
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Eight pitfalls and one solution
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Pitfall seven: False negative results
• Control experiments are necessary to tell whether the absence of a PCR product can be
interpreted as a negative result or an unsuccessful PCR
• Full proof of the significance of each PCR result can only be achieved by adding a positive control
to each single reaction
The QIAGEN OneStep Ahead RT-PCR kit’s solution:
• Duplex PCR optimized
• Enable co-amplification of an internal positive control with every single reaction
PCR1 PCR2 PCR3
With positive control
PCR1: Shows both bands = PCR worked and TOI is present
PCR2: Shows only control band = PCR worked but TOI is absent
PCR3: Shows no band = PCR did not work > repeat experiment
Without positive control
PCR1: Shows band = TOI
PCR2: No band means ?
PCR3: No band means ?
TOI = Target of interest
? ? ! !
400 bp = TOI
300 bp = internal control
PCR1 PCR2 PCR3
One Step Ahead for Your RT-PCR
22. Sample to Insight
Eight pitfalls and one solution
1. Make sure that both assays work in a singleplex reaction
2. Don‘t combine assays with amplicon lengths > 1000 bp
3. Ideally, assays don‘t differ in length by more than 200 bp
4. If one band (i.e. assay) is clearly weaker than the other, increase annealing and
extension step from 10 seconds to 20 seconds
5. If extended cycling times still don‘t give satisfying results, reduce the primer
concentration for the “stronger“ assay by increments of 0.1 µM down to 0.1 µM,
keeping the primer concentration for the “weaker“ assay at 0.5 µM
Recommendations and optimization strategies
22One Step Ahead for Your RT-PCR
23. Sample to Insight
0,1 µM -
Eight pitfalls and one solution
Effect of optimization strategies
Effect of cycling time extension
Annealing/extension: 10 s
Note: both targets (ACTB/VCL) differ
by factor >100x in mRNA abundancy
M
M
Effect of primer concentration
NTC
NTC
- 0,5 µM
- 0,5 µM
0,5 µM -
Note: both targets (PPIA/CTNNA1) differ
by >250 bp in amplicon length
Annealing/extension: 20 s
23One Step Ahead for Your RT-PCR
24. Sample to Insight
Eight pitfalls and one solution
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Pitfall eight:Target RNA
concentration threshold
• Control experiments are necessary
to tell whether the absence of a PCR
product can be interpreted as a
negative result or an unsuccessful
PCR
• Adding positive control to each
single reaction providesfull proof
about the significance of each PCR
result
The QIAGEN OneStep
Ahead RT-PCRkit’s
solution:
• Chemistry optimized for highest
sensitivity
• 2.5x master mix allows for input of
more sample RNA
Indicated amounts of HeLa total RNA (in pg) w ere used as template for amplification of
GAPDH (831bp) and ACTB (295bp) in duplicate, according to the suppliers’
instructions. Green arrow s indicate specific product, red arrow s indicate primer‐dimers.
Analysis w as performed using the QIAxcel.
24One Step Ahead for Your RT-PCR
26. Sample to Insight
How to use the kit
Kit components
Ultrapure
water
OneStep Ahead
Master Mix
Template Tracer RT-Mix
Master Mix
Tracer
Q-solution
26One Step Ahead for Your RT-PCR
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How to use the kit
27
Reactionsetup for one-stepRT-PCR
27One Step Ahead for Your RT-PCR
28. Sample to Insight
How to use the kit
Ultrafast standard protocol for amplicons <1kbp
From samples to insight in just 60 mins!
Fastest results
Reverse transcription step:
OmniScript and SensiScript RTs are
activated and reverse transcription
takes place.
The reaction temperature may be
increased up to 55°C, if satisfactory
results are not obtained at 50°C.
Initial PCR heat activation
This heating step activates DNA
Polymerase blend, inactivates
Omniscript® and Sensiscript®
Reverse Transcriptases, and
denatures the cDNA template.
10 min
@50°C
5 min
@95°C
10s
Denaturation
Annealing
Extension
Final extension
RT
PCR
40 cycles
The optimal cycle number
depends on the amount of
template RNA and the
abundance of the target
transcript.
10s
10s
2 min
28One Step Ahead for Your RT-PCR
29. Sample to Insight
How to use the kit
Protocol for amplicons 1-4 kbp
Fastest results
Reverse transcription step:
OmniScript and SensiScript RTs are
activated and reverse transcription
takes place.
Initial PCR heat activation
This heating step activates DNA
Polymerase blend, inactivates
Omniscript® and Sensiscript®
Reverse Transcriptases, and
denatures the cDNA template.
15 min
@45°C
5 min
@95°C
15s
Denaturation
Annealing
Extension
(Allow 1min per kbp amplicon size)
Final extension
RT
PCR
40 cycles
The optimal cycle number
depends on the amount of
template RNA and the
abundance of the target
transcript.
15s
1-4min
5 min
29One Step Ahead for Your RT-PCR
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How to use the kit
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Comparison of cycling protocols
Supplier QIAGEN Competitor A Competitor B
Protocol
RT 10 min / 50°C 30 min / 50°C 30 min / 55°C
Taq activiation
/ template denat.
5 min /95°C 2 min /94°C 2 min /94°C
Denaturation 10 sec / 95°C 30 sec / 94°C 15 sec / 94°C
Annealing 10 sec / 55°C 30 sec / 60°C 30 sec / 60°C
Extension 10 sec / 72°C 60 sec / 72°C 60 sec / 68°C
Total runtime (incl.
ramping)
60 min 137 min 127 min
Our new kit is more than twice as fast as the competitors
One Step Ahead for Your RT-PCR
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How to use the kit
Primer Design, Concentration and
Storage
Standard RT-PCR primers
Prerequisites for successful one-step
RT-PCR include the design of optimal
primer pairs, the use of appropriate
primer concentrations and the correct
storage of primer solutions.
The QIAGEN OneStep Ahead RT-
PCR Kit is designed to be used with
gene-specific primers only. The use
of random oligomers or oligo-dT
primers is not recommended since
this will result in the amplification of
nonspecific products.
31One Step Ahead for Your RT-PCR
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Recommendations
• Enables fully automated DNA and RNAanalysis
• Ready-to-rungel cartridges
• Fast processing: 12 samples in 3 – 10 min
• Up to 96 samples per run
(unattended processingof up to twenty 96-well plates
possible)
• Sample input amounts < 0.1 µl
• Detectionlimit of 0.1 ng/µl
• High resolution of 3 – 5 bp up to 500 bp
• Digital data output
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Data analysis – QIAxcel
One Step Ahead for Your RT-PCR
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Recommendations
QIAGEN OneStep Ahead RT-PCR Kits
https://www.QIAGEN.com/de/shop/pcr/end-point-pcr-
enzymes-and-kits/one-step-rt-pcr/QIAGEN-onestep-
ahead-rt-pcr-kit
35One Step Ahead for Your RT-PCR
39. Sample to Insight
Summary
QIAGEN OneStep Ahead RT-PCR Kit provides:
• Convenient room temperature set up also enables use in automated/robotic workflows
• Significantly shorter cycling safes time
• Highest safety and piece of mind due to built in color change pipetting control
• Highest template RNA protection due to built in RNase Inhibitor
• Higher sequence accuracy and ability to amplify longer amplicons up to 4kb
• Run internal positive control in the same reaction
• Twice the amount of reactions per kit due to reduction of reaction volume
• Convenient 2.5x master mix for easy calculation
One Step Ahead for Your RT-PCR
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Contact QIAGEN
Call: 1-800-426-8157 (NA)
+49-2103-29-12400 (EU)
Dr. Bernd Willems:
bernd.willems@QIAGEN.com
Questions?
Thank You for Attending
One Step Ahead for Your RT-PCR