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.
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.
Real Time Polymerase Chain Reaction
Basics of Real Time PCR
Definition
Advantages
Principles
Instruments (Thermal Cyclers)
Useful terms
Real Time PCR Chemistry
Fluorescence Dyes
SYBR Green
EvaGreen
Melt Doctor
Fluorescence Probes
TaqMan Probe
Molecular Beacons
Scorpion Primers
SYBR Green In details
qPCR Set-Up
Assay Design
Data Analysis
Troubleshooting
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.
Real Time Polymerase Chain Reaction
Basics of Real Time PCR
Definition
Advantages
Principles
Instruments (Thermal Cyclers)
Useful terms
Real Time PCR Chemistry
Fluorescence Dyes
SYBR Green
EvaGreen
Melt Doctor
Fluorescence Probes
TaqMan Probe
Molecular Beacons
Scorpion Primers
SYBR Green In details
qPCR Set-Up
Assay Design
Data Analysis
Troubleshooting
DNA and RNA Structure
Central Dogma of Life
Protein Engineering (Brief)
Introduction to microRNA (miRNA)
History of miRNA
Biogenesis of miRNA
Conservation of miRNA
Impact of miRNA
miRNA Therapy
Conclusion
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.
REAL TIME PCR, principle of real time pcr, method for detection real time pcr, taq man probe, molecular beacons. application of real time pcr. difference between real time pcr and conventional pcr.
The change in one nucleotide in a genome is known as single nucleotide polymorphism. There are assorted types of SNPs. SNPs can be detected by several analytical techniques i.e. DNA sequencing, microchip, HPLC and oligonucleotide ligation reaction.
Polymerase chain reaction 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
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.
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.
DNA and RNA Structure
Central Dogma of Life
Protein Engineering (Brief)
Introduction to microRNA (miRNA)
History of miRNA
Biogenesis of miRNA
Conservation of miRNA
Impact of miRNA
miRNA Therapy
Conclusion
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.
REAL TIME PCR, principle of real time pcr, method for detection real time pcr, taq man probe, molecular beacons. application of real time pcr. difference between real time pcr and conventional pcr.
The change in one nucleotide in a genome is known as single nucleotide polymorphism. There are assorted types of SNPs. SNPs can be detected by several analytical techniques i.e. DNA sequencing, microchip, HPLC and oligonucleotide ligation reaction.
Polymerase chain reaction 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
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.
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.
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.
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
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.
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!
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.
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.
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.
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.
Accreditation of Research Laboratories: An Awareness CreationSolomon Abate
This presentation is prepared for the awareness creation of EIAR center directors, quality management representatives, laboratory heads and custodians. The training is given starting November 30, 2015 for five days.
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!
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
Technical Guide to Qiagen PCR Arrays - Download the GuideQIAGEN
Total RNA discovery with RT2 and miScript PCR Arrays : Explore the RNA universe - Whatever your destination within the RNA universe, QIAGEN will help you get there. The miRNeasy kits deliver pure, high-quality total RNA from a broad range of samples. The RT2 and miScript PCR arrays are a complete solution both for focused analysis of gene and microRNA expression and for validation of microarray and RNA sequencing experiments. Together with the powerful analytics tools of GeneGlobe® and QIAGEN Ingenuity® Pathway Analysis, these products give you a smooth path from your sample to high-quality results.
RT2 Profiler PCR Arrays: Pathway-focused Gene Expression Profiling with qRT-P...QIAGEN
This paper evaluates the performance of the newest technique for monitoring the expression of a panel of pathway- or disease-specific genes: the RT2 Profiler PCR Array System. The RT2 Profiler PCR Array System combines the quantitative performance of SYBR® Green real-time PCR with the multiple-gene profiling capabilities of a microarray.
The RT2 Profiler PCR Array is a 96- or 384-well plate containing RT2 qPCR Primer Assays for a set of 84 related genes, plus 5 housekeeping genes and 3 controls. The complete system includes an instrument-specific master mix and an optimized first strand synthesis kit. This paper presents experimental data showing that RT2 Profiler PCR Arrays have the sensitivity, reproducibility, and specificity expected from real-time PCR techniques. As a result, this technology brings focused gene expression profiling to any biological laboratory setting with a real-time PCR instrument.
Targeted RNAseq for Gene Expression Using Unique Molecular Indexes (UMIs): In...QIAGEN
Traditional RNA sequencing (RNA-Seq) is a powerful tool for expression profiling, but is hindered by PCR amplification bias and inaccuracy at low expressing genes. QIAseq RNA is a flexible and precise tool developed for mitigating these complications, allowing digital gene expression analysis. This in-depth webinar will cover sample requirements, experimental design, NGS platform-specific challenges and workflow for gene enrichment, library prep and sequencing. The applications of QIASeq RNA Panels in cancer research, stem cell differentiation and elucidating the effects small molecules on signaling pathways will be highlighted.
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.
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!
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.
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.
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.
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.
Global launch of the Healthy Ageing and Prevention Index 2nd wave – alongside...ILC- UK
The Healthy Ageing and Prevention Index is an online tool created by ILC that ranks countries on six metrics including, life span, health span, work span, income, environmental performance, and happiness. The Index helps us understand how well countries have adapted to longevity and inform decision makers on what must be done to maximise the economic benefits that comes with living well for longer.
Alongside the 77th World Health Assembly in Geneva on 28 May 2024, we launched the second version of our Index, allowing us to track progress and give new insights into what needs to be done to keep populations healthier for longer.
The speakers included:
Professor Orazio Schillaci, Minister of Health, Italy
Dr Hans Groth, Chairman of the Board, World Demographic & Ageing Forum
Professor Ilona Kickbusch, Founder and Chair, Global Health Centre, Geneva Graduate Institute and co-chair, World Health Summit Council
Dr Natasha Azzopardi Muscat, Director, Country Health Policies and Systems Division, World Health Organisation EURO
Dr Marta Lomazzi, Executive Manager, World Federation of Public Health Associations
Dr Shyam Bishen, Head, Centre for Health and Healthcare and Member of the Executive Committee, World Economic Forum
Dr Karin Tegmark Wisell, Director General, Public Health Agency of Sweden
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.
Telehealth Psychology Building Trust with Clients.pptxThe Harvest Clinic
Telehealth psychology is a digital approach that offers psychological services and mental health care to clients remotely, using technologies like video conferencing, phone calls, text messaging, and mobile apps for communication.
Defecation
Normal defecation begins with movement in the left colon, moving stool toward the anus. When stool reaches the rectum, the distention causes relaxation of the internal sphincter and an awareness of the need to defecate. At the time of defecation, the external sphincter relaxes, and abdominal muscles contract, increasing intrarectal pressure and forcing the stool out
The Valsalva maneuver exerts pressure to expel faeces through a voluntary contraction of the abdominal muscles while maintaining forced expiration against a closed airway. Patients with cardiovascular disease, glaucoma, increased intracranial pressure, or a new surgical wound are at greater risk for cardiac dysrhythmias and elevated blood pressure with the Valsalva maneuver and need to avoid straining to pass the stool.
Normal defecation is painless, resulting in passage of soft, formed stool
CONSTIPATION
Constipation is a symptom, not a disease. Improper diet, reduced fluid intake, lack of exercise, and certain medications can cause constipation. For example, patients receiving opiates for pain after surgery often require a stool softener or laxative to prevent constipation. The signs of constipation include infrequent bowel movements (less than every 3 days), difficulty passing stools, excessive straining, inability to defecate at will, and hard feaces
IMPACTION
Fecal impaction results from unrelieved constipation. It is a collection of hardened feces wedged in the rectum that a person cannot expel. In cases of severe impaction the mass extends up into the sigmoid colon.
DIARRHEA
Diarrhea is an increase in the number of stools and the passage of liquid, unformed feces. It is associated with disorders affecting digestion, absorption, and secretion in the GI tract. Intestinal contents pass through the small and large intestine too quickly to allow for the usual absorption of fluid and nutrients. Irritation within the colon results in increased mucus secretion. As a result, feces become watery, and the patient is unable to control the urge to defecate. Normally an anal bag is safe and effective in long-term treatment of patients with fecal incontinence at home, in hospice, or in the hospital. Fecal incontinence is expensive and a potentially dangerous condition in terms of contamination and risk of skin ulceration
HEMORRHOIDS
Hemorrhoids are dilated, engorged veins in the lining of the rectum. They are either external or internal.
FLATULENCE
As gas accumulates in the lumen of the intestines, the bowel wall stretches and distends (flatulence). It is a common cause of abdominal fullness, pain, and cramping. Normally intestinal gas escapes through the mouth (belching) or the anus (passing of flatus)
FECAL INCONTINENCE
Fecal incontinence is the inability to control passage of feces and gas from the anus. Incontinence harms a patient’s body image
PREPARATION AND GIVING OF LAXATIVESACCORDING TO POTTER AND PERRY,
An enema is the instillation of a solution into the rectum and sig
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)
Deep Leg Vein Thrombosis (DVT): Meaning, Causes, Symptoms, Treatment, and Mor...The Lifesciences Magazine
Deep Leg Vein Thrombosis occurs when a blood clot forms in one or more of the deep veins in the legs. These clots can impede blood flow, leading to severe complications.
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.
Empowering ACOs: Leveraging Quality Management Tools for MIPS and BeyondHealth Catalyst
Join us as we delve into the crucial realm of quality reporting for MSSP (Medicare Shared Savings Program) Accountable Care Organizations (ACOs).
In this session, we will explore how a robust quality management solution can empower your organization to meet regulatory requirements and improve processes for MIPS reporting and internal quality programs. Learn how our MeasureAble application enables compliance and fosters continuous improvement.
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Pubrica’s team of researchers and writers create scientific and medical research articles, which may be important resources for authors and practitioners. Pubrica medical writers assist you in creating and revising the introduction by alerting the reader to gaps in the chosen study subject. Our professionals understand the order in which the hypothesis topic is followed by the broad subject, the issue, and the backdrop.
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How many patients does case series should have In comparison to case reports.pdf
Advanced Real-Time PCR Array Technology – Coding and Noncoding RNA Expression Analysis: qPCR Technology Webinar Series Part 2
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Coding and Noncoding RNA Expression Analysis
Coding and NoncodingRNA Expression Analysis 1
Wei Cao, PhD
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Welcome to our four-part webinar series on qPCR
2
qPCR technology overview, applications, data
analysis and service solutions
• Part 1: Introduction to Real-Time PCR (Q-PCR / qPCR/ qrt-PCR)
• Part 2: Advanced Real-Time PCR Array Technology – Coding and Noncoding RNA
Expression Analysis
• Part 3: PCR Array Data Analysis Tutorial
• Part 4: Accelerate Your Discovery With QIAGEN Service Solutions for Biomarker
Research
3. Sample to Insight
Legal disclaimer
Coding & NoncodingRNA Expression Analysis 3
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.
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Gene expression regulates biology
Coding & NoncodingRNA Expression Analysis 4
All of these pathways require molecular signaling for their activity
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A complete biological story
Coding & NoncodingRNA Expression Analysis 7
Built on pathway/network analysis
Cell cycle Angiogenesis Inflammation
Pathway
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Differential gene expression analysis
Coding & NoncodingRNA Expression Analysis 8
Question:
How can you assess the expression of different mRNAs in a
sample involved in a process (e.g., angiogenesis, cell cycle, or
inflammation) and compare it across multiple conditions?
RT2 Profiler lncRNAArrays
Answer:
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QIAGEN PCR arrays for all biomedical researchers
Coding & NoncodingRNA Expression Analysis 9
RT2 Profiler PCR Arrays for 13 species powered by over 170 pathways
Cancer and apoptosis Cytokines and inflammation Development and stem cells
Apoptosis Inflammatory cytokines Stem cells
Cell cycle Th17 for inflammation WNT signaling / notch signaling
Human miRNA array Common cytokines / chemokines Terminal differentiation markers
Breast cancer and estrogen receptor Inflammasomes TGFb / BMP signaling
Tumor metastasis NF−κB signaling pathway Endothelial cell biology
Epithelial-to-mesenchymal transition Th1-Th2-Th3 Osteogenesis
Angiogenesis TNF ligands Growth factors
Cancer drug resistance Toll-like receptors ECM and adhesion
Signal Transduction Toxicology and Drug Metabolism Neuroscience
Signal Transduction PathwayFinder Drug metabolism / drug transporters Neuroscience ion channels
NF−κB Signaling Drug phase I enzymes Neurotransmitter receptors
JAK−STAT Signaling Molecular Toxicology PathwayFinder 384HT Neurotrophins and receptors
DNA damage signaling Oxidative stress Neurogenesis and neural stem cell
Insulin signaling Stress and toxicity Parkinson’s disease
MAP kinase signaling Other Diseases Custom PCR Arrays (H/M/R/Q/D/F/P/B)
cAMP / calcium Signaling Atherosclerosis 96-well, 384-well plates
p53 Signaling Diabetes 100-well Disc, 96 × 96 chip
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QIAGEN PCR arrays for all biomedical researchers
Coding & NoncodingRNA Expression Analysis 10
The New GeneGlobe − your new research companion! Click “Browse by Research Area”
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Apply your PCR arrays4
Agenda
11
Introduction to PCR arrays1
How PCR arrays work2
PCR array performance3
What’s next?5
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Characteristics of a good qPCR Assay
Coding & NoncodingRNA Expression Analysis 12
• Amplification efficiency: exponential phase is 100%
efficient
• Sensitivity: able to detect as few as 10–50 copies of
template in one reaction
• Specificity: one assay, one target (no off-target
amplification or primer dimers)
Validation of qPCR assays should be done each
time primers are synthesized to ensure reliable performance
Can be the rate limiting step for using qPCR on 100 genes
and as a routine analysis method for a limited number of samples
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Primer assays
Coding & NoncodingRNA Expression Analysis 13
Laboratory verificationdata:
• Melting / dissociationcurve
o Single peak
• Amplification plot
o PCR efficiency
If you are designing your own primers – stop!
Every primer is experimentallyverified
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Anatomy of an RT2 Profiler PCR Array
Coding & NoncodingRNA Expression Analysis 14
• Rows A–G hold your 84 pathway-specific
genes of interest
• Five housekeeping genes
• Genomic DNA contamination control
(GDC)
• Three reverse transcription controls
(RTCS)
• Three positive PCR controls (PPCs)
• Customizable – Add four genes to a
catalog RT2 Profiler PCR Array or
completely customize based on your
research
Each well contains lyophilized, verified qPCR assays
ACTB, B2M, GAPDH, HPRT1, RPL0
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qPCR primer assays and custom PCR arrays
Coding & NoncodingRNA Expression Analysis 15
Extensive species coverage
Cow (Bos taurus) Chicken (Gallus gallus)
Horse (Equus ferus caballus) Zebrafish (Danio rerio)
Dog (Canis lupus familiaris) Pig (Sus scrofa)
Fruit fly (Drosophila melanogaster) Rabbit (Oryctolagus cuniculus)
Chinese hamster ovary (CHO) cells
(Cricetulus griseus)
Rhesus macaque (Macaca mulatta)
https://www.qiagen.com/us/products/gene and pathways/species-portal/
Wet-bench-verifiedassays for all genes in:
• Human (H / HS)
• Mouse (M / MM)
• Rat (R / RN)
At GeneGlobe, click “Browse by Species”
CustomPCR arrays are expandableto the following species:
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How are genes on PCR arrays selected?
Coding & NoncodingRNA Expression Analysis 16
PCR Array contents are relevant
Biologically relevant gene content
• Not simply biochemical pathways or kinase
cascades
✓ Published association with the biological or disease
pathways gathered from overlapping sources
including:
o Multiple publicly accessible databases
o Text mining relevant literature
Technically relevant gene content
o Use genes that are regulated at the RNA level
o Specific feedback from thought leaders
Genes in signaling pathways
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Functional gene grouping
Coding & NoncodingRNA Expression Analysis 18
Inflammatory cytokines and receptors
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Relevant pathway content on PCR arrays
Coding & NoncodingRNA Expression Analysis 19
Inflammatory cytokines and receptors
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Relevant pathway content on PCR arrays
Coding & NoncodingRNA Expression Analysis 20
Inflammatory cytokines and receptors
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Search by gene, pathway or biology
Coding & NoncodingRNA Expression Analysis 21
https://www.qiagen.com/us/geneglobe/QIAGEN GeneGlobe
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Apply your PCR arrays4
Agenda
22
Introduction to PCR arrays1
How PCR arrays work2
PCR array performance3
What’s next?5
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Principles of qPCR: an overview
Coding & NoncodingRNA Expression Analysis 23
Real-time PCR – amplify and simultaneously quantify target DNA
• qPCR (reverse transcription real-time PCR)
o Amplify and simultaneously quantify mRNA
• Ct values: threshold cycle
DNA template
(ss or ds)
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Anatomy of an RT2 Profiler PCR Array
Coding & NoncodingRNA Expression Analysis 24
• Rows A–G hold your 84 pathway-specific
genes of interest
• Five housekeeping genes
• Genomic DNA contamination control (GDC)
• Three reverse transcription controls (RTCS)
• Three positive PCR controls (PPCs)
• Customizable – Add four genes to a
catalog RT2 Profiler PCR Array or
completely customize based on your
research
Each well contains lyophilized, verified qPCR assays
ACTB, B2M, GAPDH, HPRT1, RPL0
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How RT2 Profiler PCR Arrays work
Coding & NoncodingRNA Expression Analysis 25
Experimental workflow
Stimulate cells Isolate RNA
qPCR
Convert total RNA to
cDNA
Treat cells, e.g., non-cancer
or cancer cells
Isolate RNA (RNeasy Kits)
RNase-free DNase treatment
Genomic DNA removal step
Reverse transcription step
5 min + 20 min
SYBR Green Mastermix
Real-time PCR detection
2 hr
Control Sample
Data analysis
Upload raw data (Ct) and analyze data
15 min
Data interpretation
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Interpretation of Ct values
Coding & NoncodingRNA Expression Analysis 26
Fold-change calculations for PCR array results/ data
Fold change results per gene = 2-∆∆Ct
Ex: My gene is present 4x more in the treated sample than in control sample.
Now not only one gene, but genes in a pathway (some more, some less)
Free complete and easy data analysis with web-based software
1. Raw CT value – relative expression level of that gene in the sample
2. Data normalization = ∆Ct per gene = Ctexperiment
– Ctcontrol
3. Upregulation / downregulation = ∆∆Ct per gene = ∆Ctexperiment – ∆Ctcontrol
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RT2 Profiler PCR Array data analysis
Coding & NoncodingRNA Expression Analysis 27
Free complete and easy analysis with Microsoft Excel or web-based software
• Multiple analysis formats support raw Ct analysis, fold change results, and more
Volcano plotScatter plot Clustergram
Two samples
Attend our “PCR Array Data Analysis Tutorial” webinar
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Compatibilityof QIAGEN PCR arrays
Coding & NoncodingRNA Expression Analysis 28
Use QIAGEN PCR arrays with all sample types
Option Sample types Product Cat. no.
1 Cells and easy-to-lyse tissues RNeasy Plus Mini Kit (50) 74134
2
Cells and tissues
with miRNA
miRNeasy Mini Kit (50) 217004
3 Difficult-to-lyse tissues RNeasy Plus Universal Mini Kit (50) 73404
4 Human blood PAXgene Blood RNA Kit (50) 762164
5 Animal blood RNeasy Protect Animal Blood Kit (50) 73224
6 Cells and easy-to-lyse tissues AllPrep DNA/RNA Mini Kit (50) 80204
7 Cells and all tissues AllPrep DNA/RNA/miRNA Universal Mini Kit (50) 80224
8 FFPE samples
RNeasy FFPE Kit (50) 73504
miRNeasy FFPE Kit (50) 217504
9
Exosome-derived RNA from serum
/ plasma
exoRNeasy Serum/Plasma Maxi Kit (50) 77064
Notes:
Option 3: A phenol-free alternative for fibrous tissues only: RNeasy Fibrous Tissue Mini Kit (50) (cat. no. 74704).
Accessory product: Rnase-Free DNase Set (50) (cat. no. 79254)
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Compatibilityof QIAGEN PCR arrays
Coding & NoncodingRNA Expression Analysis 29
All qPCR instruments, 96-, 100-, 384- and 96x96-well formats
Manufacturer Instruments*
Applied Biosystems • 96-well blocks: 7000, 7300, 7500, 7700, 7900HT,ViiA 7, QuantStudio
• FAST 96-well blocks: 7500, 7900HT, Step One Plus, ViiA 7, QuantStudio
• FAST 384-well blocks: 7900HT,ViiA 7, QuantStudio
Bio-Rad • iCycler, MyiQ, iQ5, CFX96, CFX384, CFX Connect
• Opticon, Opticon 2, Chromo4
Eppendorf Mastercycler ep realplex 2 / 2S / 4 / 4S
Fluidigm BioMark
QIAGEN Rotor-Gene Q, Rotor-Gene 6000
Roche LightCycler 480, LightCyler 96
Stratagene Mx3000p, Mx3005p, Mx4000p
TaKaRa TP-800 / TP-900 (Dice)
qPCR Results are independent of your PCR instrument. Your results can be replicated!
We provide service! Contact BRC.Service@qiagen.com
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Apply your PCR arrays4
Agenda
30
Introduction to PCR arrays1
How PCR arrays work2
PCR array performance3
What’s next?5
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Performance of PCR arrays
Coding & NoncodingRNA Expression Analysis 31
Every individual qPCR assayis wet-bench-verified for:
• Sensitivity (25 ng – 5 µg)
o Enhanced to <1 ng with RT2 PreAMP technology
o Compatible with WTAtechnology – down to single cells!
• Specificity
• Reproducibility
• Amplification efficiency
• Wide dynamic range
Reproducibility among different users and different instruments
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Uniformly high amplification efficiency(AE)
Coding & NoncodingRNA Expression Analysis 32
Why does this matter?
Cycle
Gene 1: Your design
(AE=100%)
Gene 1: Your colleague’s design
(AE=85%)
0 1 1
1 2 1.85
2 4 3.42
…
35 3.4x1010 2.2 x109
Δ 15x difference
This differencedoes not reflect biology — it is a technical issue
However, you would have reported it as a biological finding!
Conclusion: Using PCR arrays with uniformly high amplification efficiency, accurate
and reliable ΔCt values are guaranteed
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Replicates: technical and biological
Coding & NoncodingRNA Expression Analysis 33
Technical
• Not necessary – save time and money
• Reproducibility of PCR arrays is very high
• Results demonstrate that what you are seeing is a result of biology, not technique
• RTC and PPC show technical reproducibility on each plate, and comparable results
across plates
Biological
• Needed to verify the results are a result of biology
• At least three replicates per condition for statistical analysis (p values)
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Apply your PCR arrays4
Agenda
34
Introduction to PCR arrays1
How PCR arrays work2
PCR array performance3
What’s next?5
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Application one: apoptosis resistance
Coding & NoncodingRNA Expression Analysis 35
rhTRAIL
Most cells apoptotic
Few cells resistant to apoptosis
Cancer cells
Translating gene expression into pathways leads to molecular mechanisms
Experimental setup and researchgoals:
• What gene expression changes occur in resistant cells?
• How are these gene changes driving apoptosis resistance?
• Can this explain the mechanism of apoptosis resistance?
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Application one: experimental setup
Coding & NoncodingRNA Expression Analysis 36
• Use QIAGEN sample prep for RNA purification
(recommend miRNeasy kits for mRNA/lncRNA
and future miRNA)
• Use RT2 First Strand Kit for cDNA conversion
o Integrated DNase step
o Proprietary spike in RNA
o Priming with both oligo-dTs and
random hexamers
• Use RT2 SYBR Green master mix for qPCR
with RT2 Profiler PCR Array (RT2 Profiler
Apoptosis PCR Array (384-well))
• Go to QIAGEN’s Data Analysis Center for
analyzing lncRNA expression data
Separate
beads
Experimental setup MDA-MB-231cells
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Application one: results
Coding & NoncodingRNA Expression Analysis 37
Apoptosis
• Caspases and regulators
• Anti-apoptosis
• Pro-apoptosis
• TNF and TNFR domains
• BCL2 and BAG domains
• BIR domain
• CARD domain
• DEATH domain
• TRAF domain
One sample per plate, 370 genes
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Application one: results
Coding & NoncodingRNA Expression Analysis 38
High rhTRAIL
dose
Low rhTRAIL
dose
MDA-MB-231 cells
rhTRAIL resistant
MDA-MB-231 cells:
• Highly sensitive to TRAIL induced apoptosis at optimal TRAIL concentration
• Low doses of TRAIL result in in TRAIL-induced apoptosis-resistant cells
Results:
qPCR reveals up regulation of c-FLIP, Stat5a and Stat5b, Bcl-xL, and cyclin D1
• c-FLIP antagonizes caspase-8 activation
• Stat5b is responsible for Bcl-xL and cyclin D1 transcription
• Bcl-xL is an anti-apoptosis protein
Apoptosis
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Application two: tumor analysis
Coding & NoncodingRNA Expression Analysis 39
Many questions are raised by this experiment:
• What if you knocked down all the upregulated
genes and induced all the downregulated genes?
o Would the cells revert to normal?
• Why are TIMP3 and MMP3 upregulated
together?
o And why at different rates?
• What correlation might there be between MMP3
and MMP9?
The extracellular matrix and adhesion molecules:
• RT2 Profiler PCR Array revealed up- and
downregulated genes in breast cancer
Spend your time analyzing and
thinking about your data!
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Introduction to long non-coding RNAs (lncRNAs)
Coding & NoncodingRNA Expression Analysis 40
Long non-coding RNAs(lncRNAs)
• lncRNAs are a novel class of RNAs >200 nucleotides in size
• Many lncRNAs are molecularly indistinguishable frommRNAs
• lncRNAs regulate protein-codinggene transcription in complex ways
• Changes in lncRNAcan be correlated with a variety of human
diseases
• Most lncRNAs are localized in the nucleus with some found in the
cytoplasm
• Abundance: Some lncRNAs (e.g., MALAT1) are highly abundant
transcripts, but many lncRNAs do show a low count. Low transcription
levels do not necessarily reflect a lack of functionality
• May have a poly-Atail like mRNA
• lncRNAs tend to be less conserved acrossspecies, often showinglow
expression levels and high tissue specificity
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Expanding the RNA Universe! RT2 lncRNA qPCR system
Coding & NoncodingRNA Expression Analysis 42
• lncRNAdatabases: the in-house database at QIAGEN GeneGlobe
provides over 28,000 human and 16,000 mouse lncRNAtargets
• RT2 lncRNAassays: assays laboratory-verified for optimal qPCR
performance — high specificity, amplification efficiencyand sensitivity.
• RT2 lncRNAqPCR Arrays: pathway- or disease-relevant lncRNA
assays
• Custom option: flexible custom design from the lncRNAdatabase and
qPCR database to profile mRNA and lncRNAsimultanously
• lncRNAisolation: miRNeasy kits or exoRNeasy kits
• Data analysis: free online data anlysis tool
http://www.qiagen.com/us/landing-pages/lncrna/
lncRNA research is now possible for any lab with RNA or cDNA
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Application three: lncRNAs in prostate cancer
Coding & NoncodingRNA Expression Analysis 43
• Prostate cancer is the most common cancer in American men
• Estimated new cases in 2014: 233,000
• Estimated deaths in 2014: 29,480
• Stages
o Prostate cancer stage I: Found in the prostate only. Stage I prostate
cancer is microscopic; it can’t be felt on a digital rectal exam (DRE),
and it isn’t seen on imaging of the prostate
o Prostate cancer stage II: the tumor has grown inside the prostate but
hasn’t extended beyond it
o Prostate cancer stage III: Prostate cancer has spread outside the
prostate, but only barely. Prostate cancer in stage III may involve
nearby tissues, like the seminal vesicles
o Prostate cancer stage IV: The cancer has spread (metastasized)
outside the prostate to other tissues. Stage IV prostate cancer
commonly spreads to lymph nodes, the bones, liver or lungs
http://www.qiagen.com/us/landing-pages/lncrna/
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Application three: lncRNAs in prostate cancer
Coding & NoncodingRNA Expression Analysis 44
• Key questions:
o Can lncRNA be detected with RT2 lncRNA PCR
Arrays?
o Will literature-verifiedlncRNA expressionchange be
confirmed with RT2 lncRNAArray?
o Will there be any new links of lncRNA to prostate
cancer?
o Will there be any stage-relatedlncRNA changes
that have potential as biomarker?
http://www.qiagen.com/us/landing-pages/lncrna/
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Application three: materials and methods
Coding & NoncodingRNA Expression Analysis 45
• Samples (total RNA from fresh frozen tissue sample, reverse
transcription with RT2 First strand kit):
o Control Group: Normal control samples
o Group 1: Prostate cancer Stage II
o Group 2: Advanced Prostate cancer – Stage III-IV
• PCR Array-RT2 lncRNA PCR Array-Human Cancer
PathwayFinder (84 cancer related lncRNA assay with
controls in 96-well plate).
• Master mix: RT2 SYBR Green ROX™ qPCR Mastermix
• qPCR cyclers: ABI 7900HT
• Data analysis: GeneGlobe Data Analysis Center
http://www.qiagen.com/us/landing-pages/lncrna/
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Application three: lncRNAs in prostate cancer
Coding & NoncodingRNA Expression Analysis 47
RT2 lncRNACancer PathwayFinderPCR Array provides an easy approach for lncRNA profiling. In
prostate cancer samples the well known biomarker PCA3 was easily identified. At the same time,
novel tumor suppressor ADAMTS9-AS2 was also linked to prostate cancer progression.
Group one (prostate cancer (PCa) stage II) vs. control group (healthy controls)
Prostate cancer samples
showed significant
lncRNAexpression
changes compared with
normal controls
ADAMTS9-AS2
PCA3
Down Up
5-fold up-regulation
P value <0.01
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Application three: significant changes that correlate with PCa stage III/IV
Coding & NoncodingRNA Expression Analysis 48
By quickly screening more samples, the novel tumor suppressorlncRNAADAMTS9-AS2 was shown
to be further downregulated in stage III/IV prostate cancer samples
Is ADAMTS9-AS2 a new biomarker?More samples are needed
ADAMTS9-AS2 was shown to be further downregulated in stage III/IV
prostate cancer samples
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Apply your PCR arrays4
Agenda
49
Introduction to PCR arrays1
How PCR arrays work2
PCR array performance3
What’s next?5
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What comes after RT2 Profiler PCR Arrays?
Coding & NoncodingRNA Expression Analysis 50
• Verify results with more samples and a focusedset of genes
o RT2 qPCR Primer Assays or RT2 qPCR lncRNAPrimer Assays
o RT2 Profiler Custom PCR Arrays
• miRNA studies
o miScript miRNA PCR Arrays and qPCR Assays
• Assess biological impact
o Cignal Reporter Assays and Cignal Finder Reporter Arrays
• Quantify secretedproteins in blood, plasma and sera
o ELISArrays
• Knockdown analysis
o SureSilencing shRNAPlasmids and FlexiTube and FlexiPlate siRNA
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RT2 Profiler PCR Array system summary
Coding & NoncodingRNA Expression Analysis 51
• Complete experimental systems
o RNA isolation kits, PCR arrays, master-mix, first strand kit and free data analysis software
o Systematic controls to monitor sample quality, reverse transcription and PCR efficiency
o RNA to data analysis in only two hours
o Compatible with RNA-seq and microarray experiments but more focused
o Generate statistics – p values help justify significance of small gene expression changes
• Breadth of pathway content
o Profile the genes and pathways that you really care about
o Over 150 pathways for human, mouse, rat, Drosophila, rhesus macaque, dog, pig and more
o Customizable by gene, set or pathway
• Laboratory-verifiedqPCR performance on every lot of qPCR assays
o Amplification efficiency + specificity + high sensitivity = Reliability
• Applications by pathway, disease or expand upon single gene experiments
52. Sample to Insight
Thank you for attending!
Coding & NoncodingRNA Expression Analysis 52
Thank you for attending today’s webinar!
Contact QIAGEN
Call: 1-800-426-8157
Email: BRCsupport@QIAGEN.com
Wei Cao, Ph.D.
Wei.Cao@QIAGEN.com
Questions?
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.