This document discusses using qPCR arrays to screen and validate induced pluripotent stem cells (iPSCs). It describes how iPSCs are created by reprogramming somatic cells, and the need to validate pluripotency. Validation methods discussed include checking for pluripotency biomarkers using qPCR arrays, which allow screening multiple genes from multiple samples simultaneously. The document provides an example of a qBiomarker iPSC screening PCR array, which contains assays for 8 predictive pluripotency biomarkers, a normalization gene, and control wells to screen 8 samples per plate for pluripotent stem cell characterization.
The document discusses using PCR arrays to profile gene expression and epigenetics. PCR arrays allow researchers to analyze expression of up to 84 genes related to a pathway or disease using real-time PCR. They include controls to check for genomic DNA contamination and assay performance. As an example, the document describes how a researcher could use a PCR array to compare gene expression between metastatic and non-metastatic breast tumor samples.
New Technology and Workflow for Integrated Collection, Stabilization and Puri...QIAGEN
Research into non-invasive prenatal testing (NIPT) and circulating tumor DNA (ctDNA) testing based on circulating cell-free DNA (ccfDNA) is rapidly expanding. However, detection and quantification of ccfDNA is compromised by the release of genomic DNA (gDNA) from lymphocytes due to mechanical lysis or apoptosis during blood collection, storage and transport. PreAnalytiX has developed the PAXgene® Blood ccfDNA System, consisting of the PAXgene Blood ccfDNA Tube, a plastic blood collection tube with a unique, non-crosslinking chemistry that preserves extracellular levels of ccfDNA and prevents the release of intracellular DNA from cells into the plasma, and the QIAsymphony® PAXgene Blood ccfDNA Kit for automated ccfDNA extraction from up to 5 ml of plasma. In this webinar, this new technology development is presented in comparison to other existing technologies.
New technology and workflow for integrated collection, stabilization and puri...QIAGEN
Research into non-invasive prenatal testing (NIPT) and circulating tumor DNA (ctDNA) testing based on circulating cell-free DNA (ccfDNA) is rapidly expanding. However, detection and quantification of ccfDNA is compromised by the release of genomic DNA (gDNA) from lymphocytes due to mechanical lysis or apoptosis during blood collection, storage and transport. PreAnalytiX has developed the PAXgene Blood ccfDNA System, consisting of the PAXgene Blood ccfDNA Tube, a plastic blood collection tube with a unique, non-crosslinking chemistry that preserves extracellular levels of ccfDNA and prevents the release of intracellular DNA from cells into the plasma, and the QIAsymphonyPAXgene Blood ccfDNA Kit for automated ccfDNA extraction from up to 5 ml of plasma. In this slidedeck, this new technology development is presented in comparison to other existing technologies.
Analyzing Fusion Genes Using Next-Generation SequencingQIAGEN
Fusion genes are hybrid genes formed by the fusion of two separate genes. Translocation, interstitial deletion and chromosomal inversions are some of the genetic events that can lead to the formation of fusion genes. The occurrence of fusion genes and its implications in cancer have already been known, but the emergence of NGS technology – especially RNA sequencing – offers the potential to detect novel gene fusions. You can learn more about fusion genes and applying NGS to detect them at our upcoming webinar, presented by Raed Samara, Ph.D., QIAGEN’s Global Product Manager for NGS technologies.
In this webinar, Dr. Raed Samara will cover:
1. Fusion genes: what they are and a historical perspective
2. Fusion gene detection: the current status
3. RNA sequencing vs. digital RNA sequencing
4. How to detect and accurately quantify novel fusion genes in your sample
Extending miRQC’s dynamic range: amplifying the view of Limiting RNA samples ...QIAGEN
The original microRNA quality control (miRQC) study provided an in-depth analysis of commercially available microRNA (miRNA) quantification platforms. Specifically, twelve different
microarray, real-time PCR and small RNA sequencing platforms were assessed for reproducibility, sensitivity, accuracy, specificity and concordance of differential expression using a variety of sample types. Overall, each platform exhibited specific strengths and weaknesses, leading to the
final suggestion that a platform should be chosen on the basis of the experimental setting and the specific research questions. With this suggestion in mind, and the fact that liquid miRNA biopsies are an area of intense interest, we sought to expand the original miRQC study. For our “miRQC extension,” we benchmarked the QIAGEN miScript® PCR System with and without preamplification, and included a specific focus on routinely used biofluids. Concurrently, we benchmarked the miScript PCR System against another SYBR® Green miRNA detection platform. Overall, QIAGEN miScript demonstrated strong reproducibility and accuracy as well as superior detection rate and sensitivity in biofluids. Collectively, QIAGEN miScript provides the leading solution for novel miRNA discoveries.
Digital RNAseq Technology Introduction: Digital RNAseq Webinar Part 1QIAGEN
QIAseq RNA is a revolutionary turnkey solution for digital gene expression analysis by NGS. From 10 genes to 1000, from one sample to 100, QIAseq RNA delivers precise results on both ION and Illumina sequencing platforms. The data from QIAseq RNA is directly comparable to expression analysis derived from whole transcriptome sequencing or by qRTPCR, only better, cheaper, faster, and more flexible. This webinar will describe the principles of digital expression analysis by NGS, and review the features and benefits of the QIAseq system, options available, and the integrated data analysis package.
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.
Deeper Insight into Transcriptomes! Download the FlyerQIAGEN
Discover a new workflow for RT-PCR-based gene expression work
Accurate and biologically relevant results in RT-PCR-based
gene expression can be difficult to achieve. Successful
transcriptome work requires validated, reproducible targets
and high-quality technology. Recognizing the variability arising from sample physiology
and pathology, the influence of sample purification and
assay conditions, and the importance of access to easyto-
use software, QIAGEN experts developed a new gene
expression workflow. It will help you properly validate your
RT-PCR and gain the deepest insight into your result.
The document discusses using PCR arrays to profile gene expression and epigenetics. PCR arrays allow researchers to analyze expression of up to 84 genes related to a pathway or disease using real-time PCR. They include controls to check for genomic DNA contamination and assay performance. As an example, the document describes how a researcher could use a PCR array to compare gene expression between metastatic and non-metastatic breast tumor samples.
New Technology and Workflow for Integrated Collection, Stabilization and Puri...QIAGEN
Research into non-invasive prenatal testing (NIPT) and circulating tumor DNA (ctDNA) testing based on circulating cell-free DNA (ccfDNA) is rapidly expanding. However, detection and quantification of ccfDNA is compromised by the release of genomic DNA (gDNA) from lymphocytes due to mechanical lysis or apoptosis during blood collection, storage and transport. PreAnalytiX has developed the PAXgene® Blood ccfDNA System, consisting of the PAXgene Blood ccfDNA Tube, a plastic blood collection tube with a unique, non-crosslinking chemistry that preserves extracellular levels of ccfDNA and prevents the release of intracellular DNA from cells into the plasma, and the QIAsymphony® PAXgene Blood ccfDNA Kit for automated ccfDNA extraction from up to 5 ml of plasma. In this webinar, this new technology development is presented in comparison to other existing technologies.
New technology and workflow for integrated collection, stabilization and puri...QIAGEN
Research into non-invasive prenatal testing (NIPT) and circulating tumor DNA (ctDNA) testing based on circulating cell-free DNA (ccfDNA) is rapidly expanding. However, detection and quantification of ccfDNA is compromised by the release of genomic DNA (gDNA) from lymphocytes due to mechanical lysis or apoptosis during blood collection, storage and transport. PreAnalytiX has developed the PAXgene Blood ccfDNA System, consisting of the PAXgene Blood ccfDNA Tube, a plastic blood collection tube with a unique, non-crosslinking chemistry that preserves extracellular levels of ccfDNA and prevents the release of intracellular DNA from cells into the plasma, and the QIAsymphonyPAXgene Blood ccfDNA Kit for automated ccfDNA extraction from up to 5 ml of plasma. In this slidedeck, this new technology development is presented in comparison to other existing technologies.
Analyzing Fusion Genes Using Next-Generation SequencingQIAGEN
Fusion genes are hybrid genes formed by the fusion of two separate genes. Translocation, interstitial deletion and chromosomal inversions are some of the genetic events that can lead to the formation of fusion genes. The occurrence of fusion genes and its implications in cancer have already been known, but the emergence of NGS technology – especially RNA sequencing – offers the potential to detect novel gene fusions. You can learn more about fusion genes and applying NGS to detect them at our upcoming webinar, presented by Raed Samara, Ph.D., QIAGEN’s Global Product Manager for NGS technologies.
In this webinar, Dr. Raed Samara will cover:
1. Fusion genes: what they are and a historical perspective
2. Fusion gene detection: the current status
3. RNA sequencing vs. digital RNA sequencing
4. How to detect and accurately quantify novel fusion genes in your sample
Extending miRQC’s dynamic range: amplifying the view of Limiting RNA samples ...QIAGEN
The original microRNA quality control (miRQC) study provided an in-depth analysis of commercially available microRNA (miRNA) quantification platforms. Specifically, twelve different
microarray, real-time PCR and small RNA sequencing platforms were assessed for reproducibility, sensitivity, accuracy, specificity and concordance of differential expression using a variety of sample types. Overall, each platform exhibited specific strengths and weaknesses, leading to the
final suggestion that a platform should be chosen on the basis of the experimental setting and the specific research questions. With this suggestion in mind, and the fact that liquid miRNA biopsies are an area of intense interest, we sought to expand the original miRQC study. For our “miRQC extension,” we benchmarked the QIAGEN miScript® PCR System with and without preamplification, and included a specific focus on routinely used biofluids. Concurrently, we benchmarked the miScript PCR System against another SYBR® Green miRNA detection platform. Overall, QIAGEN miScript demonstrated strong reproducibility and accuracy as well as superior detection rate and sensitivity in biofluids. Collectively, QIAGEN miScript provides the leading solution for novel miRNA discoveries.
Digital RNAseq Technology Introduction: Digital RNAseq Webinar Part 1QIAGEN
QIAseq RNA is a revolutionary turnkey solution for digital gene expression analysis by NGS. From 10 genes to 1000, from one sample to 100, QIAseq RNA delivers precise results on both ION and Illumina sequencing platforms. The data from QIAseq RNA is directly comparable to expression analysis derived from whole transcriptome sequencing or by qRTPCR, only better, cheaper, faster, and more flexible. This webinar will describe the principles of digital expression analysis by NGS, and review the features and benefits of the QIAseq system, options available, and the integrated data analysis package.
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.
Deeper Insight into Transcriptomes! Download the FlyerQIAGEN
Discover a new workflow for RT-PCR-based gene expression work
Accurate and biologically relevant results in RT-PCR-based
gene expression can be difficult to achieve. Successful
transcriptome work requires validated, reproducible targets
and high-quality technology. Recognizing the variability arising from sample physiology
and pathology, the influence of sample purification and
assay conditions, and the importance of access to easyto-
use software, QIAGEN experts developed a new gene
expression workflow. It will help you properly validate your
RT-PCR and gain the deepest insight into your result.
This document discusses using gene and miRNA expression profiling to develop biomarkers for monitoring genotoxicity. It describes:
1. Developing gene-based in vitro biomarkers for genotoxicity using RT2 Profiler PCR Arrays to analyze expression of DNA damage and p53 pathway genes in HepG2 cells exposed to genotoxic and non-genotoxic compounds. 11 genes were identified as classifiers.
2. Identifying miRNA-based in vivo biomarkers by profiling miRNA expression in mouse liver after exposure to the carcinogen ENU using miScript PCR Arrays. The mir-34 family showed temporal changes and clustering analysis identified differentially expressed miRNAs.
3. miRNA profiles have potential to serve as biomarkers for genotoxicity
Reporter assay and q pcr application 2012Elsa von Licy
This document summarizes a presentation on high-performance cell-based assay and qPCR technologies for pathway-focused research. The presentation overview discusses QIAGEN's SABiosciences portfolio, PCR arrays, Cignal and Cignal Lenti pathway reporters, and provides a summary. PCR arrays allow analysis of mRNA expression of up to 84 genes related to biological pathways in a single experiment. Cignal reporter assays use dual-luciferase reporters to study 45 signal transduction pathways. Cignal Lenti reporters use lentiviral delivery of luciferase or GFP reporters to study pathways in difficult to transfect cells like stem cells or primary cells.
Single-cell microRNA expression profiling is a challenging workflow. From cell lysis, reverse transcription, preamplificatin to real-time PCR, every step involves technical pitfalls. Therefore it is critical to have a robust system that facilitates universal cDNA synthesis and universal amplification of all miRNAs in one workflow without introducing bias. Here we present a new poster – introducing a robust real-time PCR workflow and protocol for profiling miRNA expression from a single cell and how we analyze the single cells by using the free data analysis software.
This document describes pathway-powered PCR arrays for gene expression analysis. It discusses:
- PCR arrays that analyze the expression of genes involved in biological pathways, covering sample prep through data analysis.
- Over 140 pathway-focused PCR arrays are available covering cancer, inflammation, neuroscience, and other areas.
- The PCR arrays allow analysis of mRNA expression from various sample types in a high-sensitivity and reproducible manner using real-time PCR instrumentation.
The document summarizes three studies using RT2 Profiler PCR Arrays to examine gene expression changes in toxicology, oncology, and immunology research. In toxicology, the arrays identified distinct gene expression profiles for three drugs (troglitazone, pioglitazone, rosiglitazone) in liver cells, suggesting different mechanisms for their liver toxicity effects. In oncology, arrays revealed gene expression differences between breast tumor and normal tissue. In immunology, array results correlated well with measured cytokine protein levels between stimulated and unstimulated immune cells.
"Massively parallel sequencing in forensic genetics
Dr. Walther Parson
assoc. Prof. Institute of Legal Medicine, Innsbruck, Austria
adj. Prof. Penn State University, PA, USA"
Detection and Surveillance of Antibiotic Resistance Genes From Food and Ferti...QIAGEN
One potential way to acquire antibiotic resistance genes is through the food supply chain. Both livestock and feed may
acquire antibiotic resistant bacteria via different mechanisms. Foodstuffs can be exposed to antibiotic resistant bacteria
through fertilizer originating from waste-water treatment plants. This, in addition to increasing administration of antibiotics
to livestock, can lead to food being a potential source of antibiotic resistance genes. This may lead to horizontal gene
transfer to pathogenic enteropathogens and further to drug resistance in humans. Therefore, the surveillance and prevention
of antibiotic resistance genes in food is important.
To effectively combat the spread of difficult-to-treat bacterial infections, rapid surveillance methods to detect antibiotic
resistance genes are required; in order to monitor both bacterial isolates and metagenomic samples.
Since the gut is known to act as a reservoir for antibiotic resistance genes, a small-scale research study was performed on
5 stool samples isolated from healthy human adults using an antibiotic resistance gene identification PCR array. In addition,
the diversity of antibiotic resistance genes in municipal biosolids was determined using an Antibiotic Resistance Genes
Microbial DNA qPCR Array with DNA extracted from belt-filter, press-cake sewage samples.
22 antibiotic resistance genes were identified from different resistance classifications. Further studies were performed in
beef, chicken, vegetable and pork samples. In conclusion, PCR arrays can be effective tools for detection of antibiotic
resistance genes from food samples and potential fertilizer sources.
CAP Trapper Technologies and Applications, CAP Analysis of Gene Expression (C...Laura Berry
Presented at the NGS Tech and Applications Congress: USA. To find out more, visit:
www.global-engage.com
Masayoshi Itoh is a Senior Scientist at the RIKEN Center for Life Science Technologies (CLST) and Coordinator of the RIKEN Preventive Medicine and Diagnostics Innovation Program (PMI). In this presentation Masayoshi introduces CAP trapper technologies and presents the findings of the FANTOM5 project.
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.
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.
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.
Profile Multiple Cytokines and Chemokines Simultaneously with Very High Sensi...QIAGEN
Learn how to profile multiple cytokines and chemokines simultaneously with very high sensitivity and specificity using the standard ELISA reader. Available in different formats to suit your research needs such as single-analyte, multi-analyte or custom mix-n-match format for human, mouse and rat.
Development of quality control assays for cell-based medicinal products (ISCT...Quality Assistance s.a.
Dr. Fabian Vandermeers from Quality Assistance spoke on Development of quality control assays for cell-based medicinal products at ISCT 2017 in London.
For more information on this topic, visit: http://www.quality-assistance.com/analytical-services/CBMPs
For more information on our expertise and services, visit: www.quality-assistance.com
Follow us on social media:
LinkedIn: https://www.linkedin.com/company/quality-assistance
Twitter: https://twitter.com/QA_Belgium
Facebook: https://www.facebook.com/QualityAssistanceBelgium
Google +: https://plus.google.com/103676189647965359292
Quality Assistance S.A. is a leading European Contract Research Organisation providing the pharmaceutical industry with all the analytical services required by EMA and FDA regulations for the development and marketing of innovative human medicinal products.
We assist our clients from candidate selection, through non-clinical and clinical studies, to marketing authorisation, using our state-of-the-art, product-dedicated expertise in analytical sciences.
For each customer and each project, we design customised solutions, define analytical protocols, develop and validate specific new analytical methods and perform characterisation, stability, pharmacokinetic, biomarker and immunogenicity studies as well as batch release testing, in order to evaluate the Quality, Safety and Efficacy of the given drugs.
The document discusses clinical applications of next generation sequencing (NGS), specifically a test called NIFTY (Non-Invasive Fetal TrisomY). NIFTY uses NGS and bioinformatics to analyze cell-free fetal DNA in maternal plasma to evaluate the likelihood of fetal trisomy 21, 18, and 13. Clinical validation studies showed NIFTY has a detection rate over 99.9% for these trisomies with a low false positive rate. NIFTY provides a safe, non-invasive prenatal screening alternative to invasive diagnostic tests.
Apac distributor training series 3 swift product for cancer studySwift Biosciences
This document provides an overview of Swift Biosciences' product training series for their APAC distributors on using Swift products for cancer studies. It summarizes different Swift library preparation and sequencing kits that can be used for various cancer applications, including genomic sequencing, RNA sequencing, amplicon panels, hybridization capture, and DNA methylation. The document also reviews types of mutations found in cancer, considerations for cancer clinical workflows, and provides an example of using Swift's 2S Turbo kit for targeted sequencing of formalin-fixed, paraffin-embedded tissue samples.
Massively Parallel Sequencing - integrating the Ion PGM™ sequencer into your ...Thermo Fisher Scientific
This document summarizes the integration of massively parallel sequencing (MPS) using the Ion PGMTM sequencer into a forensic laboratory. The project aims to begin transforming STR profiling to genomic technologies, add additional SNP markers in a single workflow, and enable non-human DNA testing. Initial results show sequencing of amplified STR products is possible but alignment is challenging. A custom panel of 280 targets including STRs, SNPs, and amelogenin was also tested with most targets detected across samples. Ongoing work focuses on improving sensitivity, reproducibility, and analyzing mixed samples. Implementation of MPS as a routine forensic service is estimated within 3-5 years.
Next-generation genomics: an integrative approachHong ChangBum
This document summarizes a presentation on next-generation genomics and integrative analysis. It discusses the types of genomic data available from techniques like genome sequencing, RNA sequencing, ChIP-seq, and epigenomics. It explains that integrative analysis can help annotate functional features, infer variant function, and understand gene regulation. Approaches to integration include data reduction, unsupervised clustering, and supervised Bayesian networks. Large-scale datasets can be accessed through browsers, add-ons, and standalone tools to generate novel hypotheses. Future work includes more integrated community resources with search capabilities.
Fast qPCR assay optimization and validation techniques for HTS discusses steps for designing a successful qPCR assay, including:
1) Designing primers after running a BLAST search to check for homologous sequences and secondary structures in the target cDNA that could interfere with amplification.
2) Testing primer pairs through a temperature gradient and dilution series to validate the assay's dynamic range before running it hundreds or thousands of times.
3) Choosing an amplicon size between 75-200bp ideally, though larger sizes are possible with new fast reagents. Following these steps at the beginning leads to more effective analysis later.
Multicopy reference assay (MRef) — a superior normalizer of sample input in D...QIAGEN
Copy number variations (CNVs) and alterations (CNAs) are a source of genetic diversity in humans and are often pathogenic. Numerous CNVs and CNAs are being identified with various genome analysis platforms, including array comparative genomic hybridization (aCGH), single nucleotide polymorphism (SNP) genotyping platforms, and next-generation sequencing. Independent verification of copy number changes is a critical step. Quantitative real-time PCR (qPCR) is a classic method to verify microarray copy number findings. Traditional copy number assays that use qPCR typically rely on a putative single-copy gene reference assay (e.g., RNase P or TERT) to normalize the DNA input for downstream ΔΔCT-based copy number calculation for comparison to a reference genome. When applied to cancer samples, these single-copy reference assays may no longer be a reliable indicator of DNA input due to the presence of complex chromosome composition (both in chromosome number and structure). To meet the need for an accurate DNA input normalizer, especially for heterogeneous tumor samples, QIAGEN developed a multicopy reference (MRef) assay for real-time PCR copy number analysis. This assay, in conjunction with QIAGEN’s greater than 10 million genomewide copy number assays and pathway- and disease-focused copy number PCR arrays (Figure 1), provides a successful solution for copy number analysis. This article will address the assay design considerations, development, and performance of this multicopy reference (MRef) assay.
Comparison of the Roche AMPLICOR® Human Papillomavirus (HPV) Test with the GP...guest8b7e21d
Both the Roche AMPLICOR HPV Test and the GP5+/GP6+ PCR assay can detect HPV in cervical samples. The AMPLICOR test detects 13 high-risk HPV types while GP5+/GP6+ detects both high-risk and low-risk types. Both tests performed well on samples stored in liquid cytology media or from archived tissues. They identified the same CIN2/CIN3 samples except for two discrepancies. The AMPLICOR test may have higher analytical sensitivity for low viral loads but clinical sensitivity requires more study. Both tests can be used for HPV detection in cervical screening.
This technical article describes three case studies using RT2 Profiler PCR Arrays to analyze gene expression changes in different research areas: toxicology, oncology, and immunology. In the toxicology study, liver cells were treated with three drugs known to cause liver toxicity and the PCR Array identified distinct gene expression patterns for each drug, suggesting different mechanisms of toxicity. In the oncology study, gene expression in breast tumor samples was compared to normal tissue and a common set of upregulated genes was discovered in two independent tumor samples. In the immunology study, stimulated immune cells showed good correlation between cytokine gene and protein expression levels. The article concludes the PCR Array System is a reliable and accurate tool for pathway-focused gene expression profiling across
This document describes SureFIND Transcriptome PCR Arrays, which are ready-to-use cDNA panels that can identify the miRNAs, pathways, or transcription factors that regulate gene expression. Each array contains cDNA from cells treated with different factors, such as miRNA mimics or pathway inhibitors. The document outlines an example where a Transcriptome PCR Array identified three miRNAs - miR-193b, miR-138, and miR-373 - that regulate the INPPL1 gene. Users are encouraged to validate top hits from the arrays.
This document discusses using gene and miRNA expression profiling to develop biomarkers for monitoring genotoxicity. It describes:
1. Developing gene-based in vitro biomarkers for genotoxicity using RT2 Profiler PCR Arrays to analyze expression of DNA damage and p53 pathway genes in HepG2 cells exposed to genotoxic and non-genotoxic compounds. 11 genes were identified as classifiers.
2. Identifying miRNA-based in vivo biomarkers by profiling miRNA expression in mouse liver after exposure to the carcinogen ENU using miScript PCR Arrays. The mir-34 family showed temporal changes and clustering analysis identified differentially expressed miRNAs.
3. miRNA profiles have potential to serve as biomarkers for genotoxicity
Reporter assay and q pcr application 2012Elsa von Licy
This document summarizes a presentation on high-performance cell-based assay and qPCR technologies for pathway-focused research. The presentation overview discusses QIAGEN's SABiosciences portfolio, PCR arrays, Cignal and Cignal Lenti pathway reporters, and provides a summary. PCR arrays allow analysis of mRNA expression of up to 84 genes related to biological pathways in a single experiment. Cignal reporter assays use dual-luciferase reporters to study 45 signal transduction pathways. Cignal Lenti reporters use lentiviral delivery of luciferase or GFP reporters to study pathways in difficult to transfect cells like stem cells or primary cells.
Single-cell microRNA expression profiling is a challenging workflow. From cell lysis, reverse transcription, preamplificatin to real-time PCR, every step involves technical pitfalls. Therefore it is critical to have a robust system that facilitates universal cDNA synthesis and universal amplification of all miRNAs in one workflow without introducing bias. Here we present a new poster – introducing a robust real-time PCR workflow and protocol for profiling miRNA expression from a single cell and how we analyze the single cells by using the free data analysis software.
This document describes pathway-powered PCR arrays for gene expression analysis. It discusses:
- PCR arrays that analyze the expression of genes involved in biological pathways, covering sample prep through data analysis.
- Over 140 pathway-focused PCR arrays are available covering cancer, inflammation, neuroscience, and other areas.
- The PCR arrays allow analysis of mRNA expression from various sample types in a high-sensitivity and reproducible manner using real-time PCR instrumentation.
The document summarizes three studies using RT2 Profiler PCR Arrays to examine gene expression changes in toxicology, oncology, and immunology research. In toxicology, the arrays identified distinct gene expression profiles for three drugs (troglitazone, pioglitazone, rosiglitazone) in liver cells, suggesting different mechanisms for their liver toxicity effects. In oncology, arrays revealed gene expression differences between breast tumor and normal tissue. In immunology, array results correlated well with measured cytokine protein levels between stimulated and unstimulated immune cells.
"Massively parallel sequencing in forensic genetics
Dr. Walther Parson
assoc. Prof. Institute of Legal Medicine, Innsbruck, Austria
adj. Prof. Penn State University, PA, USA"
Detection and Surveillance of Antibiotic Resistance Genes From Food and Ferti...QIAGEN
One potential way to acquire antibiotic resistance genes is through the food supply chain. Both livestock and feed may
acquire antibiotic resistant bacteria via different mechanisms. Foodstuffs can be exposed to antibiotic resistant bacteria
through fertilizer originating from waste-water treatment plants. This, in addition to increasing administration of antibiotics
to livestock, can lead to food being a potential source of antibiotic resistance genes. This may lead to horizontal gene
transfer to pathogenic enteropathogens and further to drug resistance in humans. Therefore, the surveillance and prevention
of antibiotic resistance genes in food is important.
To effectively combat the spread of difficult-to-treat bacterial infections, rapid surveillance methods to detect antibiotic
resistance genes are required; in order to monitor both bacterial isolates and metagenomic samples.
Since the gut is known to act as a reservoir for antibiotic resistance genes, a small-scale research study was performed on
5 stool samples isolated from healthy human adults using an antibiotic resistance gene identification PCR array. In addition,
the diversity of antibiotic resistance genes in municipal biosolids was determined using an Antibiotic Resistance Genes
Microbial DNA qPCR Array with DNA extracted from belt-filter, press-cake sewage samples.
22 antibiotic resistance genes were identified from different resistance classifications. Further studies were performed in
beef, chicken, vegetable and pork samples. In conclusion, PCR arrays can be effective tools for detection of antibiotic
resistance genes from food samples and potential fertilizer sources.
CAP Trapper Technologies and Applications, CAP Analysis of Gene Expression (C...Laura Berry
Presented at the NGS Tech and Applications Congress: USA. To find out more, visit:
www.global-engage.com
Masayoshi Itoh is a Senior Scientist at the RIKEN Center for Life Science Technologies (CLST) and Coordinator of the RIKEN Preventive Medicine and Diagnostics Innovation Program (PMI). In this presentation Masayoshi introduces CAP trapper technologies and presents the findings of the FANTOM5 project.
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.
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.
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.
Profile Multiple Cytokines and Chemokines Simultaneously with Very High Sensi...QIAGEN
Learn how to profile multiple cytokines and chemokines simultaneously with very high sensitivity and specificity using the standard ELISA reader. Available in different formats to suit your research needs such as single-analyte, multi-analyte or custom mix-n-match format for human, mouse and rat.
Development of quality control assays for cell-based medicinal products (ISCT...Quality Assistance s.a.
Dr. Fabian Vandermeers from Quality Assistance spoke on Development of quality control assays for cell-based medicinal products at ISCT 2017 in London.
For more information on this topic, visit: http://www.quality-assistance.com/analytical-services/CBMPs
For more information on our expertise and services, visit: www.quality-assistance.com
Follow us on social media:
LinkedIn: https://www.linkedin.com/company/quality-assistance
Twitter: https://twitter.com/QA_Belgium
Facebook: https://www.facebook.com/QualityAssistanceBelgium
Google +: https://plus.google.com/103676189647965359292
Quality Assistance S.A. is a leading European Contract Research Organisation providing the pharmaceutical industry with all the analytical services required by EMA and FDA regulations for the development and marketing of innovative human medicinal products.
We assist our clients from candidate selection, through non-clinical and clinical studies, to marketing authorisation, using our state-of-the-art, product-dedicated expertise in analytical sciences.
For each customer and each project, we design customised solutions, define analytical protocols, develop and validate specific new analytical methods and perform characterisation, stability, pharmacokinetic, biomarker and immunogenicity studies as well as batch release testing, in order to evaluate the Quality, Safety and Efficacy of the given drugs.
The document discusses clinical applications of next generation sequencing (NGS), specifically a test called NIFTY (Non-Invasive Fetal TrisomY). NIFTY uses NGS and bioinformatics to analyze cell-free fetal DNA in maternal plasma to evaluate the likelihood of fetal trisomy 21, 18, and 13. Clinical validation studies showed NIFTY has a detection rate over 99.9% for these trisomies with a low false positive rate. NIFTY provides a safe, non-invasive prenatal screening alternative to invasive diagnostic tests.
Apac distributor training series 3 swift product for cancer studySwift Biosciences
This document provides an overview of Swift Biosciences' product training series for their APAC distributors on using Swift products for cancer studies. It summarizes different Swift library preparation and sequencing kits that can be used for various cancer applications, including genomic sequencing, RNA sequencing, amplicon panels, hybridization capture, and DNA methylation. The document also reviews types of mutations found in cancer, considerations for cancer clinical workflows, and provides an example of using Swift's 2S Turbo kit for targeted sequencing of formalin-fixed, paraffin-embedded tissue samples.
Massively Parallel Sequencing - integrating the Ion PGM™ sequencer into your ...Thermo Fisher Scientific
This document summarizes the integration of massively parallel sequencing (MPS) using the Ion PGMTM sequencer into a forensic laboratory. The project aims to begin transforming STR profiling to genomic technologies, add additional SNP markers in a single workflow, and enable non-human DNA testing. Initial results show sequencing of amplified STR products is possible but alignment is challenging. A custom panel of 280 targets including STRs, SNPs, and amelogenin was also tested with most targets detected across samples. Ongoing work focuses on improving sensitivity, reproducibility, and analyzing mixed samples. Implementation of MPS as a routine forensic service is estimated within 3-5 years.
Next-generation genomics: an integrative approachHong ChangBum
This document summarizes a presentation on next-generation genomics and integrative analysis. It discusses the types of genomic data available from techniques like genome sequencing, RNA sequencing, ChIP-seq, and epigenomics. It explains that integrative analysis can help annotate functional features, infer variant function, and understand gene regulation. Approaches to integration include data reduction, unsupervised clustering, and supervised Bayesian networks. Large-scale datasets can be accessed through browsers, add-ons, and standalone tools to generate novel hypotheses. Future work includes more integrated community resources with search capabilities.
Fast qPCR assay optimization and validation techniques for HTS discusses steps for designing a successful qPCR assay, including:
1) Designing primers after running a BLAST search to check for homologous sequences and secondary structures in the target cDNA that could interfere with amplification.
2) Testing primer pairs through a temperature gradient and dilution series to validate the assay's dynamic range before running it hundreds or thousands of times.
3) Choosing an amplicon size between 75-200bp ideally, though larger sizes are possible with new fast reagents. Following these steps at the beginning leads to more effective analysis later.
Multicopy reference assay (MRef) — a superior normalizer of sample input in D...QIAGEN
Copy number variations (CNVs) and alterations (CNAs) are a source of genetic diversity in humans and are often pathogenic. Numerous CNVs and CNAs are being identified with various genome analysis platforms, including array comparative genomic hybridization (aCGH), single nucleotide polymorphism (SNP) genotyping platforms, and next-generation sequencing. Independent verification of copy number changes is a critical step. Quantitative real-time PCR (qPCR) is a classic method to verify microarray copy number findings. Traditional copy number assays that use qPCR typically rely on a putative single-copy gene reference assay (e.g., RNase P or TERT) to normalize the DNA input for downstream ΔΔCT-based copy number calculation for comparison to a reference genome. When applied to cancer samples, these single-copy reference assays may no longer be a reliable indicator of DNA input due to the presence of complex chromosome composition (both in chromosome number and structure). To meet the need for an accurate DNA input normalizer, especially for heterogeneous tumor samples, QIAGEN developed a multicopy reference (MRef) assay for real-time PCR copy number analysis. This assay, in conjunction with QIAGEN’s greater than 10 million genomewide copy number assays and pathway- and disease-focused copy number PCR arrays (Figure 1), provides a successful solution for copy number analysis. This article will address the assay design considerations, development, and performance of this multicopy reference (MRef) assay.
Comparison of the Roche AMPLICOR® Human Papillomavirus (HPV) Test with the GP...guest8b7e21d
Both the Roche AMPLICOR HPV Test and the GP5+/GP6+ PCR assay can detect HPV in cervical samples. The AMPLICOR test detects 13 high-risk HPV types while GP5+/GP6+ detects both high-risk and low-risk types. Both tests performed well on samples stored in liquid cytology media or from archived tissues. They identified the same CIN2/CIN3 samples except for two discrepancies. The AMPLICOR test may have higher analytical sensitivity for low viral loads but clinical sensitivity requires more study. Both tests can be used for HPV detection in cervical screening.
This technical article describes three case studies using RT2 Profiler PCR Arrays to analyze gene expression changes in different research areas: toxicology, oncology, and immunology. In the toxicology study, liver cells were treated with three drugs known to cause liver toxicity and the PCR Array identified distinct gene expression patterns for each drug, suggesting different mechanisms of toxicity. In the oncology study, gene expression in breast tumor samples was compared to normal tissue and a common set of upregulated genes was discovered in two independent tumor samples. In the immunology study, stimulated immune cells showed good correlation between cytokine gene and protein expression levels. The article concludes the PCR Array System is a reliable and accurate tool for pathway-focused gene expression profiling across
This document describes SureFIND Transcriptome PCR Arrays, which are ready-to-use cDNA panels that can identify the miRNAs, pathways, or transcription factors that regulate gene expression. Each array contains cDNA from cells treated with different factors, such as miRNA mimics or pathway inhibitors. The document outlines an example where a Transcriptome PCR Array identified three miRNAs - miR-193b, miR-138, and miR-373 - that regulate the INPPL1 gene. Users are encouraged to validate top hits from the arrays.
The document describes the EpiTect ChIP qPCR System, a complete solution for chromatin immunoprecipitation (ChIP) followed by quantitative PCR (qPCR) analysis. The system includes optimized kits and reagents to simplify the multi-step ChIP workflow from chromatin extraction and immunoprecipitation to qPCR detection and data analysis. Key components are the EpiTect One-Day ChIP kit for streamlining the ChIP protocol, validated ChIP-grade antibody kits, EpiTect qPCR arrays containing pre-designed primers targeting promoter regions, and software for analyzing qPCR data. The system aims to remove technical challenges and allow researchers to focus on biological questions regarding protein-DNA interactions and epigenetic gene regulation
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.
WEBINAR Characterisation of human pluripotent stem cells (ESCs and IPSC) and ...Quality Assistance s.a.
Valérie DEFFONTAINE, R&D Scientist, Quality Assistance
Webinar held on 8th June 2017.
The discovery of human pluripotent stem cells 10 years ago turned the spotlight on the potential of pluripotent stem cells for personalised cell therapy. The scientific interest then quickly shifted towards the use of these cells for safety pharmacology, drug discovery and disease modelling. For all these purposes, in the mid to long term, properly characterised cell banks will be necessary.
The characterisation of embryonic (ESC) and induced pluripotent stem cells (IPSC) used for manufacturing requires the development and validation of analytical methods (e.g. flow cytometry, microscopy, QPCR and bioassays). Cell characterisation includes the testing of cell product identity, determination of impurities, and assessment of biological activity and viability. Among the techniques available, flow cytometry is widely used to assess the expression of cell markers. Our laboratory has developed flow cytometry panels dedicated to the characterisation of extracellular and intracellular markers of ESC and IPSC, and to the detection of cell-related impurities. We proposed a method for the validation of flow cytometry panels according to the recommendations of international guidelines on the validation of analytical methods.
IPSC differentiated into cardiomyocytes and MSC-like cells were also used to test the performance of our flow cytometry panels to accurately monitor the manufacturing process of cell products.
In addition to the technical tips, this webinar aims at presenting a critical view on the use of flow cytometry platform for cell characterisation.
For more information, visit http://www.quality-assistance.com/analytical-services/CBMPs
Polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it to a large enough amount to study in detail. PCR was invented in 1984 by the American biochemist Kary Mullis at Cetus Corporation. It is fundamental to much of genetic testing including analysis of ancient samples of DNA and identification of infectious agents. Using PCR, copies of very small amounts of DNA sequences are exponentially amplified in a series of cycles of temperature changes. PCR is now a common and often indispensable technique used in medical laboratory and clinical laboratory research for a broad variety of applications including biomedical research and criminal forensics
This document discusses new assays for microRNA (miRNA) research, including isolation, expression analysis, and functional analysis. It describes miRNA isolation kits that can purify miRNAs from various sample types. For expression analysis, it highlights real-time PCR-based miRNA assays, including miRNA PCR arrays that can profile hundreds of miRNAs simultaneously. It also discusses tools for identifying miRNA targets and analyzing miRNA function, such as miRNA mimics and inhibitors. Examples are given of how these assays have been used to study miRNAs in cancer and other diseases.
The document describes miScript miRNA PCR Arrays for analyzing miRNA expression patterns. It discusses miRNA biogenesis and function, and how the miScript system allows for genome-wide and pathway-focused miRNA analysis using a qPCR-based approach. The miScript arrays offer high reproducibility, sensitivity, and the ability to discover cancer-related and developmentally regulated miRNAs. They can be used to screen focused miRNA panels or conduct genome-wide screens to discover novel miRNA roles.
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.
qBiomarker Somatic Mutation PCR Arrays are panels of real-time PCR assays that allow for sensitive detection of mutations in 85-370 genes from fresh or FFPE samples. They provide detection of cancer-associated mutations with superior sensitivity compared to other methods using a simple real-time PCR protocol. The document describes the workflow which involves extracting DNA from samples, mixing with mastermix, distributing across the PCR array plate, running on a real-time PCR instrument, and analyzing data to make mutation calls. Examples of available arrays are provided that focus on different cancer types and pathways.
This document discusses GeneRead DNAseq Targeted Exon Enrichment and the GeneRead Library Quantification System for next generation sequencing. It begins with an introduction and agenda, then discusses targeted enrichment including the workflow, principles, data analysis, pathway content, performance data, and an application example. It also discusses library quantification including the workflow and an application example. In summary, the document presents Qiagen's GeneRead DNAseq and Library Quant systems as targeted enrichment and library quantification solutions for next generation sequencing applications.
The study of the complete set of RNAs (transcriptome) encoded by the genome of a specific cell or organism at a specific time or under a specific set of conditions is called Transcriptomics.
Transcriptomics aims:
I. To catalogue all species of transcripts, including mRNAs, noncoding RNAs and small RNAs.
II. To determine the transcriptional structure of genes, in terms of their start sites, 5′ and 3′ ends, splicing patterns and other post-transcriptional modifications.
III. To quantify the changing expression levels of each transcript during development and under different conditions.
RNA profiling is a powerful technique for understanding cellular origins and disease states. Recent studies in a variety of diseases have revealed RNA signatures that are excellent biomarker candidates for understanding disease status and predicting progression.
Suppose you want to discover a biomarker. What are the major steps in discovering a biomarker when you start from a blood sample? Here is the story of a researcher who is trying to find blood-based biomarkers in autism spectrum disorders.
A field-deployable RT-PCR system performs equivalently to real-time RT-PCR in...Simon Chung - genereach
A field-deployable RT-PCR system was found to perform equivalently to real-time RT-PCR in detecting type 2 porcine reproductive and respiratory syndrome virus (PRRSV). The field-deployable system provided results within 2 hours compared to 2.5 days for laboratory RT-PCR. Testing 50 vaccinated and 50 unvaccinated piglets over 11 weeks showed 96.25% agreement between the two methods. The field-deployable PCR system has potential for timely PRRSV detection and biosecurity management at points of need.
The document is a reference guide for pathway maps and related QIAGEN products. It includes a table of contents listing over 40 signaling pathways. For each pathway, it lists related RT2 Profiler PCR Arrays and Cignal Reporter Assay Kits from QIAGEN that can be used to study gene expression and transcription factor activity for that pathway. It also provides brief descriptions of the PCR array and reporter assay products and directs readers to QIAGEN websites for more information.
The document discusses marker-assisted breeding and the services provided by the Sequencing and Genotyping Platform. It outlines the steps in marker-assisted selection, from laying out seedlings and collecting samples to running analyses. It also lists the facilities and equipment available, including robotic platforms for liquid handling and DNA/RNA extraction, real-time PCR systems, capillary sequencers, and Illumina platforms for high-throughput genotyping. The platform provides support for marker-assisted breeding programs through services like whole genome sequencing, targeted resequencing, and protocol development for next-generation sequencing applications.
Meeting the challenges of miRNA research: miRNA and its Role in Human Disease...QIAGEN
This document discusses a 4-part webinar series on microRNA (miRNA) research presented by QIAGEN. Part 1 will cover miRNA profiling from biofluids, part 2 will discuss challenges in miRNA research, part 3 will focus on advanced miRNA expression analysis, and part 4 will analyze functional analysis of miRNA. The document provides background on miRNAs and their role in gene expression and disease. It also describes QIAGEN products and solutions for miRNA sample preparation, real-time PCR, data analysis, and functional validation to help researchers overcome challenges in miRNA analysis.
The document describes a microarray study to analyze gene expression in atherosclerotic plaques and correlate it with factors related to plaque vulnerability. Specimens will be obtained from human carotid/coronary arteries and atherosclerotic plaques in mouse models. Gene expression will be profiled using microarrays and correlated with histopathology, pH, temperature, spectroscopy and other variables. The goal is to identify genes associated with vulnerable plaques and rupture. Plaques from influenza-infected and drug-treated mice will also be analyzed to study effects on gene expression and plaque structure.
132 gene expression in atherosclerotic plaquesSHAPE Society
This document discusses microarray studies to analyze gene expression in atherosclerotic plaques and correlate it with factors related to plaque vulnerability. It begins with background on the history and applications of DNA microarrays. Key steps discussed include probe design, sample preparation including tissue collection, labeling RNA samples, hybridizing samples to a microarray chip, scanning and analyzing image data. The document outlines creating a custom microarray based on selected genes and correlating gene expression with temperature, pH, spectroscopy and histopathology of plaques. It will also analyze gene expression in influenza-infected mice and mice where plaques are induced to rupture with drugs. Human carotid artery specimens from surgery will be analyzed from symptomatic and asymptomatic patients.
The document describes a microarray study to analyze gene expression in atherosclerotic plaques and correlate it with factors related to plaque vulnerability. Specimens will be obtained from human carotid/coronary arteries and atherosclerotic plaques in mouse models. Gene expression will be profiled using microarrays and correlated with histopathology, pH, temperature, spectroscopy and other variables. Plaques from influenza-infected and drug-treated mice will also be analyzed to identify genes associated with plaque rupture. The goal is to better understand plaque vulnerability and identify potential drug targets.
Styles of Scientific Reasoning, Scientific Practices and Argument in Science ...Elsa von Licy
The document discusses various topics related to scientific reasoning, practices, and argumentation including different styles of scientific thinking, features of scientific knowledge, and teaching and learning science. It provides examples of "crazy ideas" in science that are now accepted, examines the role of argument in science, and outlines the scientific practices and central questions of science. It also discusses developing models, planning investigations, analyzing data, and constructing explanations as key scientific practices.
Anti-philosophy rejects traditional philosophy and logic, instead embracing creativity, spirituality, and personality. It considers philosophy to be dead, kept alive artificially by analytic philosophers. The document criticizes how philosophy is currently taught and argues it has become unproductive, replacing original aims with nonsense. Anti-philosophy's goal is not to destroy philosophy but to transform its current state and avoid fundamentalism in philosophy and science.
There is no_such_thing_as_a_social_science_introElsa von Licy
This document provides an introduction and overview of the arguments made in the book "There is No Such Thing as Social Science". It begins by stating the provocative title and questioning whether the authors will take it back or qualify their position.
It then outlines three ways the term "social science" could be used - referring to a scientific spirit of inquiry, a shared scientific method, or reducibility to natural sciences. The authors argue against the latter two, methodological and substantive reductionism.
The introduction discusses how opponents may accuse the authors of being a priori or anti-reductionist, but argues that those defending social science are actually being dogmatic by insisting it must follow a scientific model. It frames the debate as being
1. Reprogramming, Screening and Validation of iPSCs and
Terminally Differentiated Cells using the
qBiomarker PCR Array System
Sample & Assay Technologies
2. Outline of Webinar
What are induced pluripotent Stem Cells (iPS Cells or iPSCs)?
Methods for creating iPSCs
Screening and Validation iPSCs using qPCR
The qBiomarker PCR Array System
Application Data
Screening of iPSC for Pluripotency
Summary, Questions and Answers
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Sample & Assay Technologies
3. Why iPSCs? What can they be used for?
iPSCs are derived from somatic cells and are not subjected to moral/policy issues
that surround embryonic stem cells.
Not necessary to destroy or create embryos
Not subjected to Federal/State/Political funding issues
Uses of iPSCs in Biomedical Research:
Basic Research into pluripotency and differentiation
Applied Research into Disease Specific model systems
Translational Research Uses in regenerative medicine
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Sample & Assay Technologies
4. What are inducible pluripotent Stem Cells (iPS or iPSCs?)
Most common technique for iPSCs Creation
Reprogram a somatic cell by the over expression of key regulatory factors:
Yamanaka Factors:
Oct4, Sox2, KLF4, c-MYC
Thomson Factors:
Oct4, Sox2, Nanog, Lin28
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Sample & Assay Technologies
8. Basic Workflow in creating iPSCs
Factors:
Sox 2, Oct4, KLF4, c-Myc, Lin 28, Nanog
siRNA, small molecules, ???
Cell types:
Fibrobalsts
Pancreatic beta cells
Hepatocytes
Any somatic cell?
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Sample & Assay Technologies
9. Basic Workflow in creating iPSCs
Factors:
Sox 2, Oct4, KLF4, c-Myc, Lin 28, Nanog
siRNA, small molecules, ???
Reprogramming (2-3 weeks)
Cell types:
Fibrobalsts
Pancreatic beta cells
Hepatocytes
Any somatic cell?
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Sample & Assay Technologies
10. Basic Workflow in creating iPSCs
Factors:
Sox 2, Oct4, KLF4, c-Myc, Lin 28, Nanog
siRNA, small molecules, ???
Reprogramming (2-3 weeks)
Cell types:
Fibrobalsts
Pancreatic beta cells
Hepatocytes
Any somatic cell?
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Sample & Assay Technologies
11. Basic Workflow in creating iPSCs
Factors:
Sox 2, Oct4, KLF4, c-Myc, Lin 28, Nanog
siRNA, small molecules, ???
Delivery Methods:
Infection (lentivirus)
Transfection
Electroporation
Reprogramming (2-3 weeks)
Cell types:
Fibrobalsts
Pancreatic beta cells
Hepatocytes
Any somatic cell?
How do we validate
Pluripotency?
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Sample & Assay Technologies
12. Pluripotency Validation
How Can we validate the pluripotency of our iPSCs?
Chimeria Formation
Teratoma Formation
Biochemical Assays
?
?
?
Differentiation and Morphology (Microscopy)
Biomarkers using qPCR
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Sample & Assay Technologies
13. Pluripotency Validation
How Can we validate the pluripotency of our iPSCs?
Chimeria Formation
Teratoma Formation
Biochemical Assays
?
?
?
Differentiation and Morphology (Microscopy)
Biomarkers using qPCR
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Sample & Assay Technologies
14. Using qPCR As a Screening and Validation Technology
qPCR: (Real-Time PCR)
Able to accurately identify and provide quantitation for nucleic acids
Small amounts of sample needed
Can look at multiple genes simultaneously
SABiosciences’ Solution to qPCR is the PCR Array
Collection of Wet-Bench Validated SYBR Green qPCR Assays
Biological Content for understanding biological pathway or defining
mechanism of action
Standardized for any qPCR instrument
Data Analysis and Biological Interpretation
qBiomarker PCR Arrays use laboratory validated samples to define both a predictive
Biomarker set of genes and data analysis algorithm to clearly differentiate between a
Biological process or define disease.
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Sample & Assay Technologies
24. qBiomarker Screening and Validation PCR Arrays
2 different formats depending on number of Biomarkers
qBiomarker Screening PCR Arrays
8 samples/plate
iPSC Colony Screening
Yamanaka Factors
Thomson Factors
qBiomarker Validation PCR Arrays
4 samples/plate
iPSC Pluripotency Validation
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Sample & Assay Technologies
25. qBiomarker PCR Arrays Biological Content
Biomarkers are selected using multi-step process
1. Text mining and literature research
2. Large gene list developed
3. Biological Samples assayed through collaboration
4. Predicative Gene Signature based on Results
5. Companion Data Analysis Algorithm trained and tested
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Sample & Assay Technologies
26. How do qBiomarker PCR Arrays Work?
Isolate Total RNA from Your Sample
Isolate RNA using QIAGEN RNeasy
.
cDNA Synthesis (C-03 kit)
–
–
.
Genomic DNA Removal Step (5 min.)
Reverse Transcription Step (20 min.)
Load Plates
Multiple Samples per PCR Array
2 minutes with multi-channel pipet
.
Run 40 cycle qPCR Program
Standard cycling conditions for all
Real Time PCR Instruments
2 hours
.
Upload and Analyze Data (FREE)
10 minutes from Raw Ct values to
interpretation of Biology and Fold
Change results
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Sample & Assay Technologies
27. How do qBiomarker PCR Arrays Work?
Isolate Total RNA from Your Sample
Isolate RNA using QIAGEN RNeasy
.
cDNA Synthesis (C-03 kit)
–
–
.
Genomic DNA Removal Step (5 min.)
Reverse Transcription Step (20 min.)
Load Plates
Multiple Samples per PCR Array
2 minutes with multi-channel pipet
.
Run 40 cycle qPCR Program
Standard cycling conditions for all
Real Time PCR Instruments
2 hours
.
Upload and Analyze Data (FREE)
10 minutes from Raw Ct values to
interpretation of Biology and Fold
Change results
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Sample & Assay Technologies
28. How do qBiomarker PCR Arrays Work?
Isolate Total RNA from Your Sample
Isolate RNA using QIAGEN RNeasy
.
cDNA Synthesis (C-03 kit)
–
–
.
Genomic DNA Removal Step (5 min.)
Reverse Transcription Step (20 min.)
Load Plates
Multiple Samples per PCR Array
2 minutes with multi-channel pipet
.
Run 40 cycle qPCR Program
Standard cycling conditions for all
Real Time PCR Instruments
2 hours
.
Upload and Analyze Data (FREE)
10 minutes from Raw Ct values to
interpretation of Biology and Fold
Change results
- 28 -
Sample & Assay Technologies
29. How do qBiomarker PCR Arrays Work?
Isolate Total RNA from Your Sample
Isolate RNA using QIAGEN RNeasy
.
cDNA Synthesis (C-03 kit)
–
–
.
Genomic DNA Removal Step (5 min.)
Reverse Transcription Step (20 min.)
Load Plates
Multiple Samples per PCR Array
2 minutes with multi-channel pipet
.
Run 40 cycle qPCR Program
Standard cycling conditions for all
Real Time PCR Instruments
2 hours
.
Upload and Analyze Data (FREE)
10 minutes from Raw Ct values to
interpretation of Biology and Fold
Change results
- 29 -
Sample & Assay Technologies
30. How do qBiomarker PCR Arrays Work?
Isolate Total RNA from Your Sample
Isolate RNA using QIAGEN RNeasy
.
cDNA Synthesis (C-03 kit)
–
–
.
Genomic DNA Removal Step (5 min.)
Reverse Transcription Step (20 min.)
Load Plates
Multiple Samples per PCR Array
2 minutes with multi-channel pipet
.
Run 40 cycle qPCR Program
Standard cycling conditions for all
Real Time PCR Instruments
2 hours
.
Upload and Analyze Data (FREE)
10 minutes from Raw Ct values to
interpretation of Biology and Fold
Change results
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Sample & Assay Technologies
31. Melting Curve Analysis
Single Dissociation Peaks for Every Gene Assay
• Example of QC criteria for
every PCR Assay
manufactured by
SABiosciences
• Single peak dissociation
curves
• Single gel bands of
predicted size
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Sample & Assay Technologies
32. Melting Curve Analysis
Single Dissociation Peaks for Every Gene Assay
• Example of QC criteria for
every PCR Assay
manufactured by
SABiosciences
• Single peak dissociation
curves
• Single gel bands of
predicted size
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Sample & Assay Technologies
33. Melting Curve Analysis
Single Dissociation Peaks for Every Gene Assay
• Example of QC criteria for
every PCR Assay
manufactured by
SABiosciences
• Single peak dissociation
curves
• Single gel bands of
predicted size
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Sample & Assay Technologies
34. Compatible Instrumentation: 96- & 384-Well Formats
96-Well Blocks: 7000, 7300, 7500, 7700
FAST 96-Well Blocks: 7500, 7900HT
FAST 384-Well Block: 7900HT
StepOnePlus
iCycler, MyiQ, MyiQ2, iQ5, CFX96, CFX384
Opticon, Opticon 2, Chromo 4
Mastercycler ep realplex 2/2S/4/4S
LightCycler 480
TP-800
Mx3000p, Mx3005p, Mx4000p
qBiomarker PCR Arrays are guaranteed when using
qBiomarker qPCR SYBR Green MasterMix
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Sample & Assay Technologies
35. Data Analysis is Simple, Quick and Predictive
An integral part of ALL qBiomarker PCR Arrays
Each Pathway has a specific Data Analysis Template
Interprets Gene Expression Changes into Biological Process
.
Are my iPSCs Pluripotent?
What is the expression level of endogenous reprogramming
Factors? Exogenous?
Have my iPSCs undergone spontaneous differentiation?
Are my cardiomyocytes or neurons terminally differentiated?
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Sample & Assay Technologies
36. Screening iPS Cells for Pluripotency
Experiment:
Created 6 different iPS cell lines from fibroblasts following 3 weeks of
reprogramming. Which cell lines are totally pluripotent and worth using?
iPS Colony Screening
Delta Ct Values are interpreted
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Sample & Assay Technologies
37. Screening iPS Cells for Pluripotency
Experiment:
Created 6 different iPS cell lines from fibroblasts following 3 weeks of
reprogramming. Which cell lines are totally pluripotent and worth using?
iPS Colony Screening
Delta Ct Values are interpreted
- 37 -
Sample & Assay Technologies
38. Screening iPS Cells for Pluripotency
Experiment:
Created 6 different iPS cell lines from fibroblasts following 3 weeks of
reprogramming. Which cell lines are totally pluripotent and worth using?
iPS Colony Screening
Delta Ct Values are interpreted
Drop iPSC 1 and iPSC 2 from further experiments.
- 38 -
Sample & Assay Technologies
39. Example of Graphs
∆ Ct Value Norm alized by GAPDH
∆ Ct Value Norm alized by GAPDH
18
18
16
16
Fib
14
DNM T3B
12
iPSC 1
12
DPPA4
iPSC 2
10
iPSC 3
iPSC 4
8
∆ Ct
∆ Ct
14
GDF3
10
LEFTY1
NANOG
8
iPSC 5
6
iPSC 6
4
ESC
PODXL
POU5F1
4
2
6
ZFP42
2
0
0
DNM T3B
DPPA4
GDF3
LEFTY1
NANOG
PODXL
POU5F1
ZFP42
Fib
iPSC 1
iPSC 2
iPSC 3
Genes
iPSC 5
iPSC 6
ESC
Sam ples
Fold Change Comparing to Control Sample
Fold Change Comparing to Control Sample
5.0
5.0
4.5
4.5
4.0
iPSC 1
3.5
iPSC 2
3.0
iPSC 3
2.5
iPSC 4
2.0
iPSC 5
1.5
iPSC 6
1.0
ESC
F o ld ch an g e (L o g 10)
F old ch ang e (L o g10)
iPSC 4
0.5
DNMT3B
4.0
DPPA4
3.5
GDF3
3.0
LEFTY1
2.5
NANOG
2.0
PODXL
1.5
POU5F1
1.0
ZFP42
0.5
0.0
DNMT3B
DPPA4
GDF3
LEFTY1
NANOG
PODXL
POU5F1
0.0
ZFP42
iPSC 1
Genes
iPSC 2
iPSC 3
iPSC 4
iPSC 5
iPSC 6
ESC
Samples
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Sample & Assay Technologies
40. Application Data
Determining The Pluripotent Status of iPSCs:
.
One of the problems facing iPSC researchers is
spontaneous differentiation and loss of
pluripotency during culture and passage
conditions.
Are my IPSCs still pluripotent?
ESC A.1
iPSC #1
iPSC #2
IMR-90
iPSC Valdation PCR Array
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Sample & Assay Technologies
46. Summary
qBiomarker PCR Arrays
Laboratory validated performance and biological content
Available for any qPCR instrument
Companion Data Analysis for interpretation of results
Applications:
Screening or Validation of iPS Cell inductions
Validation of Cardiomyocyte and Neuronal Differentiation
Complete System for Reprogramming, Screening and Validating iPS Cells
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Sample & Assay Technologies
47. Thank you for attending our Webinar!
Questions, Comments or Suggestions?
Please type all questions into the “Questions” Box.
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Sample & Assay Technologies