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.
microRNA for Clinical Research and Tumor AnalysisBioGenex
The discovery of microRNAs [miRNAs] has been one of the defining developments in cancer biology over the past decade. miRNAs are short, single stranded 20-22 nucleotide long, non-coding RNAs that regulate gene expression and have fundamental roles in Cancer growth and metastasis. miRNAs exert their function via base pairing with complementary mRNA molecules, resulting in gene silencing via transcriptional repression or target degradation. BioGenex solved the inherent difficulties in visualizing miRNAs in spatial context by using a propriety technology to synthesize modified, high-affinity oligonucleotides, labelling miRNA probes with multiple reporter molecules and developing a fully-integrated miRNA-ISH workflow solution allowing high throughput analysis of miRNA in the spatial context.
Scientists have recently explored the amazing discovery that many cells produce thousands of much smaller RNA molecules, micro RNAs. Instance, more than 500 different micro RNAs have been found in human cells alone.
Micro RNA plays an important role in post-transcriptional gene regulation, such as RISC, and can cause interference and shut down gene activity.
Micro RNA is a form of ribonucleic acid and does not contain genetic information.
Mirna biogenesis, mechanism of action, isolation protocol, and quantification...SAIMA BARKI
The concise SlideShare presentation on the miRNA biogenesis, nomenclature, specific isolation protocols, the use of novel primers for preamplification purpose, and the comparison of different methods, qPCR based quantification along the guidance of the choice of sample and a novel technique for the different clinical samples.
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.
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.
microRNA for Clinical Research and Tumor AnalysisBioGenex
The discovery of microRNAs [miRNAs] has been one of the defining developments in cancer biology over the past decade. miRNAs are short, single stranded 20-22 nucleotide long, non-coding RNAs that regulate gene expression and have fundamental roles in Cancer growth and metastasis. miRNAs exert their function via base pairing with complementary mRNA molecules, resulting in gene silencing via transcriptional repression or target degradation. BioGenex solved the inherent difficulties in visualizing miRNAs in spatial context by using a propriety technology to synthesize modified, high-affinity oligonucleotides, labelling miRNA probes with multiple reporter molecules and developing a fully-integrated miRNA-ISH workflow solution allowing high throughput analysis of miRNA in the spatial context.
Scientists have recently explored the amazing discovery that many cells produce thousands of much smaller RNA molecules, micro RNAs. Instance, more than 500 different micro RNAs have been found in human cells alone.
Micro RNA plays an important role in post-transcriptional gene regulation, such as RISC, and can cause interference and shut down gene activity.
Micro RNA is a form of ribonucleic acid and does not contain genetic information.
Mirna biogenesis, mechanism of action, isolation protocol, and quantification...SAIMA BARKI
The concise SlideShare presentation on the miRNA biogenesis, nomenclature, specific isolation protocols, the use of novel primers for preamplification purpose, and the comparison of different methods, qPCR based quantification along the guidance of the choice of sample and a novel technique for the different clinical samples.
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.
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.
Cancer is a leading cause of death in developed countries. In this webcast Dr. Andreas Scherer will explain how personalized medicine can transform our approach to fighting this disease. He will also discuss current roadblocks and diagnostic challenges, and the pivotal role of Next Gen Sequencing to overcome these challenges.
The webcast will inform about best practices to design and implement a cancer testing pipeline: from sample preparation, to sequencing, to secondary and tertiary analysis of sequencing data. The goal is to rapidly identify clinically actionable data that allows an oncologist to quickly determine the best available treatment options.
The webcast will include demonstrations of the Golden Helix VarSeq software in the context of analyzing cancer gene panels and somatic mutations.
Eduardo Marbán, MD, PhD gives a deep dive into the complexities of regenerative cardiovascular medicine and the future directions for cell therapies.
Dr. Marbán’s lab has found several noncoding RNA (ncRNA) species, including short Y RNAs, which themselves have intriguing biological actions. The ncRNA within extracellular vesicles can either be used as they occur naturally or serve as bioinspiration for new chemical entities. The next generations of cell-free biologics (extracellular vesicles and noncoding RNAs) may provide, or even transcend, the benefits of cell therapy without the intrinsic limitations.
Key Topics Include:
- Understand the current status of cell therapy for heart disease
- Review the mechanisms whereby cells exert their therapeutic benefits
- Explore the future of RNA drugs inspired by the contents of extracellular vesicles
Liquid Biopsy Overview, Challenges and New Solutions: Liquid Biopsy Series Pa...QIAGEN
A liquid biopsy is often described as a sensitive and specific blood test to detect circulating tumor cells (CTCs). CTCs, shed by both the primary and metastasized tumors, carry specific information about their origins and markers that will enable us to discover new diagnosis, prognosis and therapeutic targets. This slidedeck gives an overview of the recent progress in exploring the predictive potential of circulating biomarkers, including circulating tumor cells, circulating tumor DNA, microRNAs, long non-coding RNAs (lncRNAs) and exosomes. Addressing both biological and technical aspects, we detail the isolation and characterization of circulating biomarkers. Challenges and solutions are also featured.
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.
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.
Cancer is a leading cause of death in developed countries. In this webcast Dr. Andreas Scherer will explain how personalized medicine can transform our approach to fighting this disease. He will also discuss current roadblocks and diagnostic challenges, and the pivotal role of Next Gen Sequencing to overcome these challenges.
The webcast will inform about best practices to design and implement a cancer testing pipeline: from sample preparation, to sequencing, to secondary and tertiary analysis of sequencing data. The goal is to rapidly identify clinically actionable data that allows an oncologist to quickly determine the best available treatment options.
The webcast will include demonstrations of the Golden Helix VarSeq software in the context of analyzing cancer gene panels and somatic mutations.
Eduardo Marbán, MD, PhD gives a deep dive into the complexities of regenerative cardiovascular medicine and the future directions for cell therapies.
Dr. Marbán’s lab has found several noncoding RNA (ncRNA) species, including short Y RNAs, which themselves have intriguing biological actions. The ncRNA within extracellular vesicles can either be used as they occur naturally or serve as bioinspiration for new chemical entities. The next generations of cell-free biologics (extracellular vesicles and noncoding RNAs) may provide, or even transcend, the benefits of cell therapy without the intrinsic limitations.
Key Topics Include:
- Understand the current status of cell therapy for heart disease
- Review the mechanisms whereby cells exert their therapeutic benefits
- Explore the future of RNA drugs inspired by the contents of extracellular vesicles
Liquid Biopsy Overview, Challenges and New Solutions: Liquid Biopsy Series Pa...QIAGEN
A liquid biopsy is often described as a sensitive and specific blood test to detect circulating tumor cells (CTCs). CTCs, shed by both the primary and metastasized tumors, carry specific information about their origins and markers that will enable us to discover new diagnosis, prognosis and therapeutic targets. This slidedeck gives an overview of the recent progress in exploring the predictive potential of circulating biomarkers, including circulating tumor cells, circulating tumor DNA, microRNAs, long non-coding RNAs (lncRNAs) and exosomes. Addressing both biological and technical aspects, we detail the isolation and characterization of circulating biomarkers. Challenges and solutions are also featured.
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.
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.
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.
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
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.
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.
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
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.
The Importance of Quality Control Steps in ExperimentsQIAGEN
From starting material to final results, every analysis workflow is a journey to unlock the biological information within your sample without altering it, and high-quality results are only achieved from high-quality samples.
Within each step, lie challenges directly related to the sample type and analysis technologies, and at each step, there is potential for multiple things to go wrong, jeopardizing your experiments, results and reputation. Therefore, standardizing samples and performing relevant quality control after critical steps is of utmost importance to ensure the quality and reproducibility of results, as well as reliable interpretation.
In this webinar, we will introduce you to the main sample quality parameters and their respective impact on downstream applications, discuss how to monitor them and cover the advantages of automating quality control along complex workflows.
Potentiality of a triple microRNA classifier: miR- 193a-3p, miR-23a and miR-3...Enrique Moreno Gonzalez
MicroRNAs (miRNAs) are short, non-coding RNA molecules that act as regulators of gene expression. Circulating blood miRNAs offer great potential as cancer biomarkers. The objective of this study was to correlate the differential expression of miRNAs in tissue and blood in the identification of biomarkers for early detection of colorectal cancer (CRC).
MicroRNA Stability in FFPE Tissue Samples: Dependence on GC Contentkalahawer
MicroRNAs (miRNAs) are small non-coding RNAs responsible for fine-tuning of gene
expression at post-transcriptional level. The alterations in miRNA expression levels profoundly
affect human health and often lead to the development of severe diseases. Currently,
high throughput analyses, such as microarray and deep sequencing, are performed
in order to identify miRNA biomarkers, using archival patient tissue samples. MiRNAs are
more robust than longer RNAs, and resistant to extreme temperatures, pH, and formalinfixed
paraffin-embedding (FFPE) process. Here, we have compared the stability of miRNAs
in FFPE cardiac tissues using next-generation sequencing. The mode read length in FFPE
samples was 11 nucleotides (nt), while that in the matched frozen samples was 22 nt.
Although the read counts were increased 1.7-fold in FFPE samples, compared with those in
the frozen samples, the average miRNA mapping rate decreased from 32.0% to 9.4%.
These results indicate that, in addition to the fragmentation of longer RNAs, miRNAs are to
some extent degraded in FFPE tissues as well. The expression profiles of total miRNAs in
two groups were highly correlated (0.88 <r < 0.92). However, the relative read count of each
miRNA was different depending on the GC content (p<0.0001). The unequal degradation of
each miRNA affected the abundance ranking in the library, and miR-133a was shown to be
the most abundant in FFPE cardiac tissues instead of miR-1, which was predominant
before fixation. Subsequent quantitative PCR (qPCR) analyses revealed that miRNAs with
GC content of less than 40% are more degraded than GC-rich miRNAs (p<0.0001).We
showed that deep sequencing data obtained using FFPE samples cannot be directly compared
with that of fresh frozen samples. The combination of miRNA deep sequencing and
other quantitative analyses, such as qPCR, may improve the utility of archival FFPE tissue
samples.
Diagonsis of cancer through saliva.pptxZaidAhmad42
Human saliva is an ideal body fluid for developing non-invasive diagnostics. Saliva contains naturally-occurring nanoparticles with unique structural and biochemical characteristics.
Forecasting clinical behavior and therapeutic response of human cancer currently utilizes a limited number of tumor markers in combination with characteristics of the patient and their disease. Although few tumor markers and molecular targets exist for evaluation, the wealth of information derived from recent sequencing advancements provides greater opportunities to develop more precise tests for diagnostics, prognostics, therapy selection and monitoring in the future. The objectives of this study are to study miRNA and mRNA expression profiles of laser capture microdissection (LCM)-procured tumor cells and intact serial sections of breast tissue samples using next generation sequencing (NGS) methods. Our hypothesis is that miRNA signatures discerned from specific tumor cell populations more precisely correlate with behavior than that provided by conventional biomarkers from intact tissue samples. Additionally, we hypothesize the data generated in this study will present mRNA signatures informative for breast tumor research and support our miRNA findings through suggesting relevant miRNA:mRNA target associations.
De-identified frozen research samples of primary invasive ductal tumors of known grade and biomarker status containing 35-70% tumor were selected from an IRB-approved Biorepository. Comparison of expressed miRNAs from intact tissue sections with those of cognate tumor cells procured by LCM revealed, in general, that smaller defined miRNA gene sets were expressed in LCM isolated populations of tumor cells. In addition to miRNA sequencing, targeted RNA sequencing with the Ion AmpliSeq™ Transcriptome Human Gene Expression Kit was used to capture mRNA expression information. Data presented here demonstrates high mapping rates for targeted mRNA (>91% of reads) and miRNA (> 88% of reads) libraries. We also demonstrate high technical reproducibility between multiple libraries from the same tumor sample for both mRNA (R>0.99) and miRNA (R>0.97) libraries. We also report suggested miRNA:mRNA target associations identified in our set of breast tumor research samples. These data provide insights into breast cancer biology that may lead to new molecular diagnostics and targets for drug design in the future as well as an improved understanding of the molecular basis of clinical behavior and potential therapeutic response.
2014 11-27 ODDP 2014 course, Amsterdam, Alain van GoolAlain van Gool
Presentation as part of a comprehensive oncology drug development course, to discuss a pharmaceutical approach to identify, validate and develop biomarkers for personalized medicine for melanoma.
Development and Commercialisation of a Molecular Diagnostic CompanyMalavikaSankararaman
Development and commercialisation of a spin-out molecular diagnostics approach to detect different grades of Prostate Cancer using Fourier Transform Infrared Spectroscopy
miRNA profiling from blood challenges and recommendations - Download the articleQIAGEN
The discovery of stable miRNA species circulating in blood has led to increased research focus on disease-related variations in serum and plasma miRNA expression and the possibility that such variations could serve as noninvasive biomarkers for disease. Working with serum and plasma miRNA presents various challenges in purification and characterization. In this paper, we outline QIAGEN recommendations for robust purification and quantification, as well as reliable data normalization and analysis.
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.
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 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.
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.
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.
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
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.
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.
How many patients does case series should have In comparison to case reports.pdfpubrica101
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.
https://pubrica.com/academy/case-study-or-series/how-many-patients-does-case-series-should-have-in-comparison-to-case-reports/
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)
CHAPTER 1 SEMESTER V - ROLE OF PEADIATRIC NURSE.pdfSachin Sharma
Pediatric nurses play a vital role in the health and well-being of children. Their responsibilities are wide-ranging, and their objectives can be categorized into several key areas:
1. Direct Patient Care:
Objective: Provide comprehensive and compassionate care to infants, children, and adolescents in various healthcare settings (hospitals, clinics, etc.).
This includes tasks like:
Monitoring vital signs and physical condition.
Administering medications and treatments.
Performing procedures as directed by doctors.
Assisting with daily living activities (bathing, feeding).
Providing emotional support and pain management.
2. Health Promotion and Education:
Objective: Promote healthy behaviors and educate children, families, and communities about preventive healthcare.
This includes tasks like:
Administering vaccinations.
Providing education on nutrition, hygiene, and development.
Offering breastfeeding and childbirth support.
Counseling families on safety and injury prevention.
3. Collaboration and Advocacy:
Objective: Collaborate effectively with doctors, social workers, therapists, and other healthcare professionals to ensure coordinated care for children.
Objective: Advocate for the rights and best interests of their patients, especially when children cannot speak for themselves.
This includes tasks like:
Communicating effectively with healthcare teams.
Identifying and addressing potential risks to child welfare.
Educating families about their child's condition and treatment options.
4. Professional Development and Research:
Objective: Stay up-to-date on the latest advancements in pediatric healthcare through continuing education and research.
Objective: Contribute to improving the quality of care for children by participating in research initiatives.
This includes tasks like:
Attending workshops and conferences on pediatric nursing.
Participating in clinical trials related to child health.
Implementing evidence-based practices into their daily routines.
By fulfilling these objectives, pediatric nurses play a crucial role in ensuring the optimal health and well-being of children throughout all stages of their development.
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.
Explore our infographic on 'Essential Metrics for Palliative Care Management' which highlights key performance indicators crucial for enhancing the quality and efficiency of palliative care services.
This visual guide breaks down important metrics across four categories: Patient-Centered Metrics, Care Efficiency Metrics, Quality of Life Metrics, and Staff Metrics. Each section is designed to help healthcare professionals monitor and improve care delivery for patients facing serious illnesses. Understand how to implement these metrics in your palliative care practices for better outcomes and higher satisfaction levels.
ICH Guidelines for Pharmacovigilance.pdfNEHA GUPTA
The "ICH Guidelines for Pharmacovigilance" PDF provides a comprehensive overview of the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines related to pharmacovigilance. These guidelines aim to ensure that drugs are safe and effective for patients by monitoring and assessing adverse effects, ensuring proper reporting systems, and improving risk management practices. The document is essential for professionals in the pharmaceutical industry, regulatory authorities, and healthcare providers, offering detailed procedures and standards for pharmacovigilance activities to enhance drug safety and protect public health.
Biofluid miRNA profiling: from sample to biomarker: miRNA and its Role in Human Disease Webinar Series Part 1
1. Sample to Insight
Biofluid miRNA profiling: from sample to biomarker
Jonathan Shaffer, Ph.D., Senior Scientist, Product Development
Jonathan.Shaffer@qiagen.com
2. Sample to Insight
Welcome to our four-part webinar series on miRNAs
2
Part 1: Biofluid miRNA profiling: from sample to biomarker
Part 2: Meeting the challenges of miRNA research
Part 3: Advanced miRNA expression analysis
Part 4: Functional analysis of miRNA
miRNA and its role in human disease
Biomarker Discovery in Biofluids: From Sample to Biomarker
3. Sample to Insight
Legal disclaimer
Biomarker Discovery in Biofluids: From Sample to Biomarker 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.
4. Sample to Insight
QIAGEN tools for biomarker discovery4
Agenda
4
Background1
What is your biomarker I.Q.?2
Sample miRNA biomarker project3
5. Sample to Insight
What is a biomarker?
Biomarker Discovery in Biofluids: From Sample to Biomarker 5
A characteristic that is objectively
measured and evaluated as an
indicator of normal biologic
processes, pathogenic processes or
pharmacologic responses to a
therapeutic intervention
Characteristic Methodology
Presence of antibodies ELISA
Abnormal bp, blood cell counts, electrolyte blood counts, pressure
Distinct histological indicators microscopy
Abnormal liver function markers biofluid assay
Presence of muscle injury protein markers biofluid assay
Elevated kidney marker: serum creatinine biofluid assay
Gene status or gene expression status qPCR, NGS, array, etc.
6. Sample to Insight
Cancer biomarker
Biomarker Discovery in Biofluids: From Sample to Biomarker 6
Personalized medicine
Is this the
optimal drug
for my
cancer?
Predictive
Is it likely to
develop this
cancer?
Prognostic
What type of
cancer is it?
Diagnostic
What’s the
optimal dose
for my
body?
Pharmacodynamics
Will the
cancer
return?
Recurrence
7. Sample to Insight
Noninvasive biomarker
Biomarker Discovery in Biofluids: From Sample to Biomarker 7
In order to use a biomarker for diagnostics, the sample must be as easy to obtain as possible
Urine or saliva sample
A drop of blood like those diabetes patients extract
Blood sample taken by a doctor
CSF
Surgical biopsy
Evaluation
8. Sample to Insight
Canonical pathway of miRNA biogenesis
Biomarker Discovery in Biofluids: From Sample to Biomarker 8
Transcribed by RNA polymerase II as a long
primary transcript (pri-miRNAs), which may contain
more than one miRNA
In the nucleus, pri-miRNAs are processed to
hairpin-like pre-miRNAs by the RNase III Drosha
Pre-miRNAs are then exported to the cytosol by
exportin 5
In the cytosol, the RNAse III Dicer processes these
precursors to mature miRNAs
These miRNAs are incorporated in RISC
miRNAs with high homology to the target mRNA
lead to mRNA cleavage
miRNAs with imperfect base pairing to the target
mRNA lead to translational repression and / or
mRNA degradation
9. Sample to Insight
Why miRNA biomarkers?
Biomarker Discovery in Biofluids: From Sample to Biomarker 9
Changes in miRNA can be correlated with gene expression changes in
development, differentiation, signal transduction, infection, aging and disease
miRNA expression analysis is the foundation for these discoveries
miRBase Entries
10. Sample to Insight
Circulating miRNA biomarkers in the press
Biomarker Discovery in Biofluids: From Sample to Biomarker 10
No shortage of candidate genes
Cho (2011) Front. Gene. 2
MicroRNA Deregulation in cancer Theragnostic and prognostic value Reference
let-7a Decrease in gastric cancer Discriminate gastric cancer from healthy controls Tsujiura et al. (2010)
let-7f Decrease in NSCLC Associated with overall survival in NSCLC Silva et al. (2011)
miR-1 Decrease in NSCLC Associated with overall survival in NSCLC Hu et al. (2010)
miR-10b Increase in breast cancer Associated with metastases in breast cancer Roth et al. (2010)
miR-17 Increase in gastric cancer Discriminate gastric cancer from healthy controls Zhou et al. (2010)
miR-17 + 106a Increase in gastric cancer Discriminate gastric cancer from healthy controls Zhou et al. (2010)
miR-17-3p Increase in CRC Discriminate CRC from healthy controls Ng et al. (2009)
miR-17-5p Increase in gastric cancer Discriminate gastric cancer from healthy controls Tsujiura et al. (2010)
miR-20b Decrease in NSCLC
Associated with advanced stages and lymph node
metastases in NSCLC
Silva et al. (2011)
miR-21
Increase in CLL harboring 17p deletion
Increase in gastric cancer
Associated with overall survival in CLL
Discriminate gastric cancer from healthy controls
Rossi et al. (2010)
Tsujiura et al. (2010)
miRs-21 + 126 + 210 + 486-5p Deregulate in NSCLC Discriminate stage I NSCLC from healthy controls Shen et al. (2011)
miRs-21 + 155 + 196a + 210 Increase in pancreatic adenocarcinoma
Discriminate pancreatic adenocarcinoma from
healthy controls
Wang et al. (2009)
11. Sample to Insight
Biomarker Discovery in Biofluids: From Sample to Biomarker 11
Analytes:
Cell-free miRNA,
mRNA, DNA
Samples:
Serum, plasma,
cerebrospinal fluid (CSF)
12. Sample to Insight
What is blood?
Biomarker Discovery in Biofluids: From Sample to Biomarker 12
RBC, WBC, platelets, CTC, “othercells”,extracellular?
RBC, WBC,
platelets, other cells
(e.g., circulating tumor cells)
Serum (post clotting)
Plasma (no-clotting)
High levels of nucleases present in plasma
Freely circulating nucleic acids should be rapidly degraded
Surprisingly, stable nucleic acids can be detected in serum and plasma
13. Sample to Insight
Stable miRNAs in circulation
Biomarker Discovery in Biofluids: From Sample to Biomarker 13
An evolving story
1) Valadi, H., et.al.,(2007) Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells, Nat Cell Biol
9:654-659
2) Hunter MP et. al., (2008) Detection of microRNA Expression in Human Peripheral Blood Microvesicles, PLoS ONE 3:e3694
3) Kosaka, N et. al (2010) Secretory mechanisms and intercellular transfer of microRNAs in living cells, J Biol Chem 285: 17442-17452
4) Arroyo, JD et. al., (2011) Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma, Proc. Natl. Acad.
Sci 108: 5003-5008
5) Vickers, KC., et. al., (2011) MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins. Nat Cell Biol 13:423
6) Wang K, Zhang S, Weber J, Baxter D, Galas DJ.(2010) Export of microRNAs and microRNA-protective protein by mammalian cells. Nucleic Acids Res.
2010 Nov 1;38(20):7248-59.
14. Sample to Insight
Exosomes / microvesicles (MVs)
Biomarker Discovery in Biofluids: From Sample to Biomarker 14
It’sthecargothatmatters
Blood Plasma
MVs/exosomes are ~50–200 nm
small vesicles excreted by all
cells
MVs/exosomes are found in all
biofluids (e.g., blood)
MVs/exosomes contain stable
RNA (mRNA, miRNA, other
small RNAs), DNA and protein
Contents are specifically
packaged
Mechanism of local and distant
cellular communication
Promise for disease detection
and monitoring
Use exosomal miRNA profiling in
the absence of tissue to
accurately reflectthetumor’s
profile
15. Sample to Insight
Should you only look at exosomes?
Biomarker Discovery in Biofluids: From Sample to Biomarker 15
No…well,sometimes!
Potentially 90% of miRNAs in circulation are present in a non-membrane-bound
form consistent with a ribonucleoprotein complex
At the same time, analyzing the exosome fraction could maximize signal-to-noise
ratio of a potential biomarker, providing better sensitivity
16. Sample to Insight
QIAGEN tools for biomarker discovery4
Agenda
16
Background1
What is your biomarker I.Q.?2
Sample miRNA biomarker project3
Isolationa
Quantificationb
17. Sample to Insight
PAXgene® Blood miRNA System
Biomarker Discovery in Biofluids: From Sample to Biomarker 17
Collect / stabilize and purify total RNA from whole blood
Collection/Stabilization: PAXgene Blood RNA Tubes
Draw volume: 2.5 ml
Preservation of cellular RNA at point of collection
Sample stabilization during transport and storage
Up to 3 days at 18–25°C
Up to 5 days at 2–8°C
Seven years storage at –20°C or –80°C (studies ongoing)
Purification: PAXgene Blood miRNA Kit
High purity RNA from PAXgene-stabilized whole blood
High yields of small RNA species
Manual and automated solutions
18. Sample to Insight
Total RNA (miRNA + mRNA) isolation from cell-free body fluids
Biomarker Discovery in Biofluids: From Sample to Biomarker 18
miRNeasy Serum / Plasma Kit
Includes synthetic RNA control assay for normalization
Minimal elution volume (14 µl)
High-purity RNA suitable for all downstream applications
Easy, robust procedures
Automatable protocol
When possible, avoid heparanized plasma
If you can‘t avoid heparinized samples, let me know! We
have a solution!
Purification of circulating RNA from
plasma, serum, CSF, saliva, urine, etc.
19. Sample to Insight
Exosome purification and total RNA isolation from serum / plasma
Biomarker Discovery in Biofluids: From Sample to Biomarker 19
exoRNeasy Serum/Plasma Maxi Kit
exoRNeasy Serum/Plasma Midi Kit
Specifically enriches for RNA contained in vesicles
Quickly isolates purified total RNA from microvesicles
Efficiently isolates mRNA and miRNA from plasma/serum
Enables use of high input volumes for sensitive detection of
low abundance transcripts
Maxi: 4 ml
Midi: 1 ml
20. Sample to Insight
Intact vesicles are eluted from the exoEasy column
Biomarker Discovery in Biofluids: From Sample to Biomarker 20
Scanning EM (20000x magnification) reveals higher purity with exoEasy
Both preparations contain vesicle-shaped structures within an expected size range
UC: Many smaller, unidentified structures/particles that do not match the expected size
exoEasy: Intact vesicles with higher purity
Ultracentrifugation (UC) Eluate from exoEasy
21. Sample to Insight
exoRNeasy Serum / Plasma Maxi Kit
Biomarker Discovery in Biofluids: From Sample to Biomarker 21
Workflow
Microvesicle
isolation
20 minutes
RNA isolation
35 minutes
Separate serum /
plasma
Isolate
exosomes
Isolate
RNA
22. Sample to Insight
QIAGEN tools for biomarker discovery4
Agenda
22
Background1
What is your biomarker I.Q.?2
Sample miRNA biomarker project3
Isolationa
Quantificationb
23. Sample to Insight
Three phase workflow: sample to classifier
Biomarker Discovery in Biofluids: From Sample to Biomarker 23
Phase I: Qualitatively determine expressed miRNAs
Pooled samples
miRNome profiling (or NGS)
Phase II: Quantitatively determine differentially expressed miRNAs
Individual profiling of samples (that went into pools)
Screen only expressed miRNAs from Phase I
Phase III: Develop and test classifier model
Addition individual profiling of samples
Screen differentially expressed miRNAs from Phase II
24. Sample to Insight
miScript PCR System
Biomarker Discovery in Biofluids: From Sample to Biomarker 24
Complete miRNA quantification system
1. miScript II RT Kit
HiFlex Buffer: Unparalleled flexibility for miRNA and
mRNA quantification from a single cDNA preparation
HiSpec Buffer: Unmatched specificity for mature
miRNA profiling
2. miScript miRNA PCR Arrays
miRNome
Pathway-focused
3. miScript PreAMP Kit
Optional step for small or precious samples
Full miRNome profiling from as little as 1 ng RNA
4. Assays
miScript Primer Assays
miScript Precursor Assays
QuantiTect Primer Assays
5. miScript SYBR Green PCR Kit
QuantiTect SYBR Green PCR Master Mix
Universal Primer
6. miScript miRNA PCR Array data analysis software
Straightforward, free data analysis
25. Sample to Insight
miScript PCR System
Biomarker Discovery in Biofluids: From Sample to Biomarker 25
Workflow
1. Isolate total RNA
2. Perform reverse transcription
3. Prepare PCR pre-mix
4. Load PCR arrays or plates
5. Perform real-time PCR
6. Analyze data
26. Sample to Insight
miRNA expression profiling: miScript miRNA PCR Arrays
Biomarker Discovery in Biofluids: From Sample to Biomarker 26
Wet-lab-verified miScript Primer Assays pre-dried in PCR plates
miRBase Profiler miRNome Arrays
Most species
Largest content
High content (HC) arrays
Targeted miRNome profiling
Focused arrays
Bioinformatic-driven profiling
Formats
96-well, 384-well, Fluidigm® BioMarkTM
Compatible with virtually all mainstream real-time instruments
Fully customizable
Prep your PCR reaction mix Load your plate Run your real-time experiment!
No pipetting of individual primers!
27. Sample to Insight
miRNA expression profiling: miScript miRNA PCR Arrays (cont.)
Biomarker Discovery in Biofluids: From Sample to Biomarker 27
miRBase Profiler miScript miRNA PCR Array
Human
Coverage through miRBase v21
2402 primer assays!
Mouse
Coverage through miRBase v21
1765 primer assays!
Rat
Dog
Rhesus macaque
Cow
100% validated assays
Each assay is bench validated
Each array is quality controlled
Leading miRNome coverage
Completely scalable!
Choose as many plates as you
want…profilethe v21 miRNome
…profileonly the v16 miRNome
Contact product development if
there is interest in other species!
miRBase Profiler Arrays Benefits of miRBase Profiler Arrays
The most complete, validated miRNome available!
28. Sample to Insight
Are newly annotated miRNAs even expressed?
Biomarker Discovery in Biofluids: From Sample to Biomarker 28
Liver tissue profiling of a pool of ten healthy male liver tissues
miScript
Vendor 2
Vendor 3
29. Sample to Insight
Limiting samples: miScript PreAMP Kit
Biomarker Discovery in Biofluids: From Sample to Biomarker 29
miRNome profiling from as little as 1 ng total RNA
Highly multiplex, PCR-based preamplification
Compatible with all miScript miRNA PCR Arrays and miScript Primer Assays
Enables miRNA profiling experiments using very limited amounts of starting material
Cell or tissues: 1 ng total RNA
Fluids:
Serum / plasma: 50 µl or less
Urine: Any amount
CSF: Any amount
Aqueous humor: Any amount
When in doubt, ‘miScript PreAMP’ it!
30. Sample to Insight
High-throughput miRNA expression profiling: miScript Microfluidics
Biomarker Discovery in Biofluids: From Sample to Biomarker 30
First complete system for miRNA expression profiling on the Fluidigm® BioMarkTM
Why use miScript on the BioMark?
96 samples, 384 assays
4 Fluidigm Real-Time PCR Chips
5 h per Chip
36,864 data points in 20 h
(only 2 days!)
31. Sample to Insight
QIAGEN tools for biomarker discovery4
Agenda
31
Background1
What is your biomarker I.Q.?2
Sample miRNA biomarker project3
32. Sample to Insight
Liver toxicity miRNA biomarker project with Dr. James Dear
Biomarker Discovery in Biofluids: From Sample to Biomarker 32
Biomarker Discovery Workflow
Acetaminophen (APAP) overdose is a common poisoning
worldwide and can cause liver damage, potentially
resulting in acute liver failure and death
In the US and UK, acetaminophen overdose is the most
common cause of acute liver failure
Scope of collaboration:
Determine miRNA markers of acetaminophen poisoning
Distinguish acetaminophen poisoning from other liver
syndromes
Experiment workflow: biomarker discovery
Phase 1: 356 expressed miRNAs
Narrowed list from 1809 miRNAs
Phase 2: 85 differentially expressed miRNAs
Phase 3: Classifier developed and tested that can
separate APAP-TOX from APAP-No TOX
33. Sample to Insight
Phase I: Qualitatively determine expressed miRNAs
Biomarker Discovery in Biofluids: From Sample to Biomarker 33
Goal: Establishthe“APAPmiRNome”byassayingpooledsampleswiththehumanmiRNome
Samples: 54 acetaminophen (APAP) overdose plasma samples
27 APAP-no TOX: acetaminophen overdose without toxicity
27 APAP-TOX: acetaminophen toxicity
Total RNA isolation: miRNeasy Serum/Plasma Kit
Random RNA pool preparation
2 APAP-no TOX pools: 9 samples per pool
2 APAP-TOX pools: 9 samples per pool
miRNA expression profiling: miScript PCR System
Human miRNome V18 (1809 bench-verified primers)
Selection: Qualitative determination
CT < limit of detection (CT = 35) with a single, sharp melt peak
How many miRNAs were selected? 356
miRNAs expressed in all 4 pools: 132
miRNAs expressed in 3 pools: 105
miRNAs expressed in 2 pools: 114
miRNAs expressed in 1 pool: 5
34. Sample to Insight
Phase II: Quantitatively determine differentially expressed miRNAs
Biomarker Discovery in Biofluids: From Sample to Biomarker 34
Goal: Determine miRNAs differentially expressed in response to APAP toxicity
Total RNA Samples: 54 acetaminophen (APAP) overdose plasma samples from Phase I
27 APAP-no TOX: acetaminophen overdose without toxicity
27 APAP-TOX: acetaminophen toxicity
miRNA expression profiling: miScript PCR System
356 human miRNAs
Fluidigm BioMark HD (4 runs)
Results:
85 differentially expressed miRNAs
± 3-fold, p-value < 0.05
Well expressed in APAP-no TOX,
APAP-TOX, or both
1 snoRNA
7 invariantly expressed miRNAs
Identified using NormFinder
Very important for data normalization
miR-122-5p
miR-885-5p
35. Sample to Insight
Phase III: Develop and test classifier model
Biomarker Discovery in Biofluids: From Sample to Biomarker 35
Goal: Develop and test a classifier that accurately separates APAP-TOX and APAP-no TOX
Step 1: Select the most predictive 16 miRNAs from training data set (93 targets)
Train random forest classifier: 250 bootstrap subsamples (80%) without replacement
Determined importance rank of each target
Performed prediction on remaining 20% of samples
Class separation magnitude and model error
250 iterations of classifier training: Determined median importance rank of each target
Selected the 16 most predictive targets
Step 2: Train a final random forest classifier using 16 most predictive miRNAs
Most predictive miRNAs
Importance Rank miRNA ID
1 - 11 miRNA #1 - #11
12 hsa-miR-122-5p
13 miRNA #13
14 hsa-miR-885-5p
15 miRNA #15
16 miRNA #16
Class Separation
36. Sample to Insight
Phase III: Develop and test classifier model (cont.)
Biomarker Discovery in Biofluids: From Sample to Biomarker 36
Goal: Develop and test a classifier that accurately separates APAP-TOX and APAP-no TOX
Step 3: Test the random forest classifier on an independent, blinded test data set (81 samples)
Sensitivity: 90%
Specificity: 92%
Classifier can efficiently separate
APAP-no TOX from APAP-TOX
37. Sample to Insight
Biomarker development
Biomarker Discovery in Biofluids: From Sample to Biomarker 37
Important considerations
Be novel! Initial whole miRNome screening = unique signatures!
If the group would have only profiled the first two plates (roughly 768 mature miRNAs):
Only 64 of 92 differentially expressed or invariant miRNAs would have been assayed
30% loss of data
Two invariant miRNAs would have been missed
Five miRNAs from optimal 16 miRNA signature would not have been assayed
Three of top five miRNAs from the signature would not have been assayed
CT value normalization is critical!
snoRNAs / snRNAs do not exhibit robust expression in cell-free biofluids and should not be
selected as normalization controls
Large assay panel normalization: CT mean of commonly expressed miRNAs
Small assay panel normalization: Invariant miRNA(s)
Verify miRNA signature on naïve samples
Strong changes might be general indicators or even non-specific
Other liver disease? Stress markers?
Relatively weak changes might add specificity
What’s next?
Revisit literature, Ingenuity® Pathway Analysis (IPA), etc.
Screen other types of samples to help define specificity and refine signature.
38. Sample to Insight
Biomarker Discovery in Biofluids: From Sample to Biomarker 38
Circulating mRNA
Biomarker Discovery
Exosomes contain stable RNA including mRNA
Isolation
exoRNeasy Serum / Plasma Maxi Kit
Reverse Transcription/PreAMP
RT2 PreAMP cDNA Synthesis Kit
RT2 PreAMP Pathway Primer Mix
Real-time PCR
RT2 Profiler PCR Arrays
RT2 qPCR Primer Assays
39. Sample to Insight
QIAGEN tools for biomarker discovery4
Agenda
39
Background1
What is your biomarker I.Q.?2
Sample miRNA biomarker project3
40. Sample to Insight
Where can I find the products discussed today?
Biomarker Discovery in Biofluids: From Sample to Biomarker 40
www.qiagen.com
www.qiagen.com/GeneGlobe
41. Sample to Insight
Sample to Insight miRNA portfolio
Biomarker Discovery in Biofluids: From Sample to Biomarker 41
miRNeasy Mini Kit, miRNeasy Micro Kit miScript II RT Kit HiPerFect Transfection Reagent
miRNeasy 96 Kit miScript Plant RT Kit Attractene Transfection Reagent
miRNeasy FFPE Kit miScript PreAMP Kit miScript miRNA Mimics
miRNeasy Serum/Plasma Kit miScript SYBR Green PCR Kit miScript miRNA Inhibitors
Modified miRNeasy Mini Kit for plant
tissues
miScript miRNA PCR Arrays Custom miScript miRNA Mimics
PAXgene Tissue miRNA Kit miScript Microfluidics for Fluidigm Mimic and inhibitor controls
PAXgene Blood miRNA Kit miScript Primer Assay miScript Target Protector
Supplementary protocol for miRNA from
Plasma and Serum
miScript Precursor Assay
miScript miRNA Inhibitor 96 and 384
Plates and Sets
Profiling
QIAcube QIAgility Rotor-Gene QQIAGEN Service Core
FunctionalizationIsolation
Quantification
and profiling
42. Sample to Insight
Sample to Insight mRNA portfolio
Biomarker Discovery in Biofluids: From Sample to Biomarker 42
Isolation
Quantification
and profiling
RNeasy Mini Kit RT2 First Strand cDNA Kits
RNeasy Microarray Tissue Mini Kit RT2 qPCR Master Mixes
RNeasy FFPE Kit RT2 Profiler PCR Arrays (Profiling)
RNeasy Micro Kit RT2 qPCR Primer Assays
PAXgene Blood RNA Kit GeneGlobe Data Analysis Center
QIAcube QIAgility Rotor-Gene QHigh-throughput
43. Sample to Insight
Biomarker Discovery in Biofluids: From Sample to Biomarker 43
Thank you for attending today’s webinar!
Jonathan Shaffer, Ph.D.
Jonathan.Shaffer@qiagen.com
Contact QIAGEN
1-800-426-8157
BRCsupport@QIAGEN.com
Questions?
Editor's Notes
PreAnalytiX is using a systems approach offering a collection device as well as a purification kit.
The PAXgene Blood RNA Tube is a plastic tube with 2.5 ml draw volume. RNA is preserved at the time point of collection and can be stored and transported at various temperatures. For shipment issues a huge benefit is the stabilization for up to 3 days at room temperature and up to 5 days at 4°C.
Furthermore, we there are ongoing studies in our laboratories where we could claim for 6-7 years stability at -20°C and -80°C.
High yields of small RNA species. Blood was collected and stored for 20 – 24 hours at room temperature in the PAXgene Blood RNA Tube before freezing at - 15 to - 30°C. RNA was purified using the PAXgene Blood RNA Kit (RNA kit) or the PAXgene Blood miRNA Kit (miRNA kit). (A) Purified RNA was analyzed by agarose gel electrophoresis