The document discusses Cignal Reporter Assays, which are cell-based assays for analyzing gene expression and signaling pathways. The assays use dual-luciferase technology and transcription factor-targeted response elements to provide sensitive and reproducible measurements of 45 signaling pathways. Key advantages of the assays include minimizing experimental variability through dual-luciferase normalization, increasing signal-to-noise ratio using destabilized and codon-optimized luciferase, and maximizing response using optimized transcriptional response elements tailored to each pathway. The assays are available in multiple formats including plasmid, lentiviral, luciferase and GFP, making them suitable for a variety of experimental systems and applications.
New Progress in Pyrosequencing for DNA MethylationQIAGEN
Pyrosequencing is a highly flexible technology that lets you rapidly analyze short- to medium-length sequences fast and quantitatively with high accuracy. The real-time, high-resolution sequence output makes the technology highly suitable for applications including complex mutation analysis, microbial identification and DNA methylation quantification.
The main bottleneck in Pyrosequencing has been limited sequence length, which is critical for some applications. Our new technology, software, and chemistry overcome this bottleneck and give sequence reads that are typically twice as long as those from previous PyroMark systems. The new PyroMark Q24 Advanced system also reduces background noise, improving quantification even at sites distant from the sequencing start. The new system is ideal for applications requiring analysis of longer sequences, such as DNA methylation analysis in epigenetic research, frequency determination in mutation analysis, and various de novo sequencing applications.
In this presentation, we will discuss the following applications and technology improvements:
• DNA methylation analysis at single base resolution at CpG and CpN sites
• Improved quantification of sequence variations at any sequence position
• Easy and improved base calling functionality
The document describes Cignal Reporter Assays from SABiosciences that enable simple and robust analysis of signal transduction pathways. The assays utilize dual-luciferase reporters containing optimized transcriptional regulatory elements and luciferase variants to provide high sensitivity and low variability. The assays allow monitoring of 29 pathways and are available in different formats for various cell types and applications like RNAi and small molecule screening.
Ambion scientists Emily Zeringer and Marie Gonzalez presented the background, methods and what to expect when extracting nucleic acids from FFPE tissue samples. These are the slides from the presentation. The presentation can be viewed with audio here http://find.lifetechnologies.com/ambion/ffpewebinar/sldshr
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
Cancer Research & the Challenges of FFPE Samples – An IntroductionQIAGEN
A cascade of complex genetic and epigenetic changes regulate tumor formation and progression. Gene expression analyses can shed light on these changes at a molecular level and identify the key genes and associated pathways involved in cancer. Often the samples used in cancer research are FFPE samples, which pose a significant challenge in terms of nucleic acid quality. The quality of nucleic acids extracted from FFPE samples depends on a number of factors, including how the samples were handled before, during and after fixation and embedding.
Dr. Vishwadeepak Tripathi describes the variability of sample purification from FFPE samples – in particular, samples to be used in cancer research. What are the challenges and solutions, and what quality control approach can ensure credible results? This webinar will focus on sample purification and the quality control of FFPE samples and compare different automated purification procedures.
The document discusses Cignal Lenti Reporter Assays for analyzing signaling pathways in mammalian cells. It describes:
1) The challenges of traditional cell-based signaling assays such as poor reproducibility, low sensitivity, and inability to study non-transfectable cell types.
2) How the Cignal Lenti Reporters address these challenges by using optimized transcriptional response elements and reporter genes engineered for high performance, and lentiviral delivery which allows transduction of any cell type.
3) The product breadth including over 45 pathways covered, dual reporter formats of luciferase and GFP, and array formats for studying multiple pathways simultaneously.
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!
RotorGene Q A Rapid, Automatable real-time PCR Instrument for Genotyping and...QIAGEN
The document describes the Rotor-Gene Q real-time PCR instrument. It has a unique centrifugal design that allows for excellent thermal and optical precision compared to block-based instruments. Each reaction tube spins individually, providing very precise temperature uniformity between wells. It also uses LED technology for excitation and filters for detection, allowing for multiple detection channels without need for normalization or reference dyes. Applications mentioned include quantitative PCR, high resolution melt analysis, genotyping, pathogen detection, and multiplex PCR. QIAGEN products are highlighted such as kits, controls, and the QIAgility liquid handler for automated real-time PCR setup.
New Progress in Pyrosequencing for DNA MethylationQIAGEN
Pyrosequencing is a highly flexible technology that lets you rapidly analyze short- to medium-length sequences fast and quantitatively with high accuracy. The real-time, high-resolution sequence output makes the technology highly suitable for applications including complex mutation analysis, microbial identification and DNA methylation quantification.
The main bottleneck in Pyrosequencing has been limited sequence length, which is critical for some applications. Our new technology, software, and chemistry overcome this bottleneck and give sequence reads that are typically twice as long as those from previous PyroMark systems. The new PyroMark Q24 Advanced system also reduces background noise, improving quantification even at sites distant from the sequencing start. The new system is ideal for applications requiring analysis of longer sequences, such as DNA methylation analysis in epigenetic research, frequency determination in mutation analysis, and various de novo sequencing applications.
In this presentation, we will discuss the following applications and technology improvements:
• DNA methylation analysis at single base resolution at CpG and CpN sites
• Improved quantification of sequence variations at any sequence position
• Easy and improved base calling functionality
The document describes Cignal Reporter Assays from SABiosciences that enable simple and robust analysis of signal transduction pathways. The assays utilize dual-luciferase reporters containing optimized transcriptional regulatory elements and luciferase variants to provide high sensitivity and low variability. The assays allow monitoring of 29 pathways and are available in different formats for various cell types and applications like RNAi and small molecule screening.
Ambion scientists Emily Zeringer and Marie Gonzalez presented the background, methods and what to expect when extracting nucleic acids from FFPE tissue samples. These are the slides from the presentation. The presentation can be viewed with audio here http://find.lifetechnologies.com/ambion/ffpewebinar/sldshr
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
Cancer Research & the Challenges of FFPE Samples – An IntroductionQIAGEN
A cascade of complex genetic and epigenetic changes regulate tumor formation and progression. Gene expression analyses can shed light on these changes at a molecular level and identify the key genes and associated pathways involved in cancer. Often the samples used in cancer research are FFPE samples, which pose a significant challenge in terms of nucleic acid quality. The quality of nucleic acids extracted from FFPE samples depends on a number of factors, including how the samples were handled before, during and after fixation and embedding.
Dr. Vishwadeepak Tripathi describes the variability of sample purification from FFPE samples – in particular, samples to be used in cancer research. What are the challenges and solutions, and what quality control approach can ensure credible results? This webinar will focus on sample purification and the quality control of FFPE samples and compare different automated purification procedures.
The document discusses Cignal Lenti Reporter Assays for analyzing signaling pathways in mammalian cells. It describes:
1) The challenges of traditional cell-based signaling assays such as poor reproducibility, low sensitivity, and inability to study non-transfectable cell types.
2) How the Cignal Lenti Reporters address these challenges by using optimized transcriptional response elements and reporter genes engineered for high performance, and lentiviral delivery which allows transduction of any cell type.
3) The product breadth including over 45 pathways covered, dual reporter formats of luciferase and GFP, and array formats for studying multiple pathways simultaneously.
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!
RotorGene Q A Rapid, Automatable real-time PCR Instrument for Genotyping and...QIAGEN
The document describes the Rotor-Gene Q real-time PCR instrument. It has a unique centrifugal design that allows for excellent thermal and optical precision compared to block-based instruments. Each reaction tube spins individually, providing very precise temperature uniformity between wells. It also uses LED technology for excitation and filters for detection, allowing for multiple detection channels without need for normalization or reference dyes. Applications mentioned include quantitative PCR, high resolution melt analysis, genotyping, pathogen detection, and multiplex PCR. QIAGEN products are highlighted such as kits, controls, and the QIAgility liquid handler for automated real-time PCR setup.
Challenges of FFPE Sample Materials – Where Does Variation in Quantity of Pur...QIAGEN
In this slidedeck, we reveal how to get the most from your FFPE samples. We discuss variability in quantity and purity of DNA purified from FFPE samples manually or with automated procedures, assessed by different quantification and quality control methods. You can also learn more about the QIAxpert system and how it can help you gain reliable quantification of FFPE samples.
PCR Array Data Analysis Tutorial: qPCR Technology Webinar Series Part 3QIAGEN
This webinar presentation provides an overview and tutorial on analyzing data from RT2 Profiler PCR Array experiments. It discusses organizing raw Ct value data, performing ΔCt and ΔΔCt calculations to analyze gene expression changes between sample groups, and using the GeneGlobe Data Analysis Center web portal to analyze the data. The webinar highlights new features of the Data Analysis Center including improved data visualization and an upgraded sample manager. It emphasizes following the standard protocol for setting baselines and thresholds when analyzing PCR array data.
Dna Methylation Analysis in a Single Day - Download the SlidesQIAGEN
This webinar introduces the new PyroMark Q48 Autoprep system. Combined with the latest EpiTect Fast bisulfite conversion technology, the new PyroMark Q48 Autoprep can now provide highly automated methylation analysis in a single day.
The document discusses eight common challenges to successful one-step RT-PCR and how the new QIAGEN OneStep Ahead RT-PCR Kit addresses each challenge. The kit contains optimized components that allow reverse transcription and PCR amplification to take place in a single tube without extra optimization. It ensures efficient, highly specific reactions with convenient room temperature setup and a visual pipetting control. The kit also offers the fastest cycling protocol on the market by completing the one-step RT-PCR in just one hour.
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.
2011 course on Molecular Diagnostic Automation - Part 2 - AmplificationPatrick Merel
2011 course on Molecular Diagnostic Automation - Part 2 - Amplification.
This is from early 2011. Prices and Specifications of instruments may have changed.
Part 2 of 3
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 document provides an overview of polymerase chain reaction (PCR) for field epidemiologists. It describes the history and invention of PCR in 1983. It explains that PCR enzymatically amplifies DNA using two primers and 30-35 cycles to exponentially replicate the target DNA sequence. The document outlines the process of PCR including denaturing, annealing and extending DNA strands. It discusses the advantages of PCR being quick, reliable and sensitive for detecting specific pathogens or genes. However, it also notes disadvantages like the need for equipment and risks of contamination producing false results. Examples of PCR applications include detecting viruses, bacteria, and analyzing antibiotic resistance genes.
Enabling RNA-Seq With Limited RNA Using Whole Transcriptome AmplificationQIAGEN
RNA-Seq was developed to perform transcriptome profiling and provides a highly precise measurement of expression levels of transcripts and their isoforms. Normally, RNA-Seq analysis requires at least 500 ng –1 μg of total RNA. When working with small biopsies, single cells (such as circulating tumor cells), or other limited material, whole transcriptome amplification (WTA) is normally required. Various WTA methods overcome limited RNA availability and enable transcriptome analysis from limited material or even single cells. In standard PCR-based WTA procedures, however, bias from uneven coverage of cDNA regions with high GC or AT content or amplification errors can lead to the loss of transcripts and wrong variant calling. Here, we compare a standard RNA-Seq library preparation method and the REPLI-g RNA library protocol. The REPLI-g procedure is a PCR-free protocol to efficiently generate RNA-Seq libraries from small amounts of RNA or a single cell in 6.5–7 hours. The REPLI-g protocol uses whole transcriptome amplification based on multiple displacement amplification (MDA), combined with an efficient library adaptor ligation procedure, to prepare RNA-Seq libraries from small RNA amounts. The procedure demonstrates high fidelity, minimal bias and retention of sample‘s transcriptional profile. Compared to standard RNA-Seq library prep, the REPLI-g protocol demonstrates similar reproducibility and sensitivity in transcript detection.
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.
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
Automated Nucleic Acid Purification from Diverse Sample types using dedicated...QIAGEN
This webinar will focus on the automation of QIAGEN’s new line of DNA and RNA sample prep kits for the microbiome. We will show how automation on the QIAcube enables efficient and reliable use of these samples for sensitive downstream applications such as qPCR and NGS. In addition, you will learn how to successfully use the CLC Microbial Genomics Module for metagenome sequencing and identification of microbial composition and diversity.
This document discusses various nucleic acid amplification tests (NAATs) for detecting Chlamydia trachomatis, including PCR, real-time PCR, transcription-mediated amplification (TMA), and strand displacement amplification (SDA). It provides details on commercially available tests like Cobas Amplicor, Roche cobas, Abbott RealTime, Gen-Probe Aptima Combo 2, and BD ProbeTec systems. The advantages and limitations of each method are outlined. Sensitivity and specificity rates for different sample types are generally high, ranging from 92-100% sensitivity and 95-100% specificity.
Single-nucleotide polymorphisms (SNPs) provide important information about the biology and evolution of different organisms. SNPs may also help predict an individual’s response to certain drugs, susceptibility to environmental factors, and risk of developing particular diseases providing valuable insight into pathophysiology of the human condition. As a result, SNPs with important functional roles often become subjects for high-throughput experiments.
In this webinar, Daniel Tsang provides an overview of genotyping using real-time PCR (qPCR) technology, including challenges and ways to overcome these challenges. He presents a novel qPCR-based genotyping solution, the rhAmp™ SNP Genotyping System, along with its advantages in genotyping, details on cluster separation, as well as solutions to improve the calling accuracy and confidence of making genotype calls.
The document discusses using NCBI databases to design quantitative PCR (qPCR) assays. It describes several NCBI tools that can be used:
1) The NCBI Nucleotide and Gene databases to obtain sequence information for the gene of interest.
2) NCBI BLAST to perform sequence searches and check primer specificity against relevant databases.
3) NCBI dbSNP to search for single nucleotide polymorphisms (SNPs) in the primer binding sites that could affect assay performance.
The document provides guidance on how to use these NCBI tools at various steps of the qPCR assay design process.
QIAcube® RNA isolation from stool samples using the RNeasy® PowerMicrobiome® ...QIAGEN
This application note demonstrates that RNA is extracted efficiently from stool samples using the RNeasy PowerMicrobiome Kit and the QIAcube system. Furthermore, the RNA isolated with the RNeasy PowerMicrobiome Kit and the QIAcube system is compatible with downstream applications.
Semi Automated Low-throughput Workflow for Microbial Analyses of Human StoolQIAGEN
The gut microbiota composition changes dramatically throughout aging and disease. A healthy gut microbiota is typically characterized by large bacterial taxonomic diversity and functional capacity, whereas frailty and aging are associated with loss of diversity and expansion of more pathogenic bacterial species. However, in order to accurately profile changes in microbial communities, the reproducible isolation of high-quality DNA is an important step. Automation enables reliable and reproducible isolation of DNA of superior quality, which can be used directly for downstream sequencing applications.
This webinar focuses on the development of a semi-automated workflow to profile the gut microbiota of young and old individuals and identify changes in bacterial composition and function that occur with age. This workflow will help to simplify and streamline the DNA extraction process for samples with high inhibitor content and subsequent microbial community analyses.
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.
RNA Integrity and Quality – Standardize RNA Quality Control QIAGEN
This document discusses RNA quality control and integrity. It emphasizes that RNA integrity is critical for obtaining accurate gene expression measurements. The RNA Integrity Number (RIN) provides a standardized score to assess RNA integrity based on capillary electrophoresis. Maintaining high RNA purity and avoiding degradation are important to ensure stable RNA samples that can be reliably stored. The QIAxpert system allows comprehensive RNA quality control by assessing concentration, purity, integrity, and contaminants in a single analysis.
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.
Site directed mutagenesis of β2-microglobulin PowerPoint PresentationTyler Liang
This document describes site-directed mutagenesis experiments performed on beta-2 microglobulin (β2m) to study its role in amyloidosis. Primers were designed to introduce mutations D39V and I5T into β2m. Polymerase chain reaction and transformation were used to generate mutant plasmids, which were sequenced to confirm the mutations. Future studies are planned to introduce mutations W61F and W96F to further study β2m stability and aggregation.
Codon bias as a means to fine tune geneAisha Hussain
This document discusses codon bias and how it can be used to fine-tune gene expression. It explains that the genetic code is degenerate, with multiple codons encoding the same amino acid. Organisms exhibit codon bias, preferring certain codons. Codon bias correlates with tRNA abundance and affects translation efficiency. Rare codons can slow translation to regulate co-translational folding and membrane translocation. Codon landscapes within genes also influence expression. Exploiting codon bias through tRNA supplementation or gene optimization improves heterologous protein production. Further integrating experimental data will advance understanding and synthetic biology applications of codon bias.
Challenges of FFPE Sample Materials – Where Does Variation in Quantity of Pur...QIAGEN
In this slidedeck, we reveal how to get the most from your FFPE samples. We discuss variability in quantity and purity of DNA purified from FFPE samples manually or with automated procedures, assessed by different quantification and quality control methods. You can also learn more about the QIAxpert system and how it can help you gain reliable quantification of FFPE samples.
PCR Array Data Analysis Tutorial: qPCR Technology Webinar Series Part 3QIAGEN
This webinar presentation provides an overview and tutorial on analyzing data from RT2 Profiler PCR Array experiments. It discusses organizing raw Ct value data, performing ΔCt and ΔΔCt calculations to analyze gene expression changes between sample groups, and using the GeneGlobe Data Analysis Center web portal to analyze the data. The webinar highlights new features of the Data Analysis Center including improved data visualization and an upgraded sample manager. It emphasizes following the standard protocol for setting baselines and thresholds when analyzing PCR array data.
Dna Methylation Analysis in a Single Day - Download the SlidesQIAGEN
This webinar introduces the new PyroMark Q48 Autoprep system. Combined with the latest EpiTect Fast bisulfite conversion technology, the new PyroMark Q48 Autoprep can now provide highly automated methylation analysis in a single day.
The document discusses eight common challenges to successful one-step RT-PCR and how the new QIAGEN OneStep Ahead RT-PCR Kit addresses each challenge. The kit contains optimized components that allow reverse transcription and PCR amplification to take place in a single tube without extra optimization. It ensures efficient, highly specific reactions with convenient room temperature setup and a visual pipetting control. The kit also offers the fastest cycling protocol on the market by completing the one-step RT-PCR in just one hour.
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.
2011 course on Molecular Diagnostic Automation - Part 2 - AmplificationPatrick Merel
2011 course on Molecular Diagnostic Automation - Part 2 - Amplification.
This is from early 2011. Prices and Specifications of instruments may have changed.
Part 2 of 3
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 document provides an overview of polymerase chain reaction (PCR) for field epidemiologists. It describes the history and invention of PCR in 1983. It explains that PCR enzymatically amplifies DNA using two primers and 30-35 cycles to exponentially replicate the target DNA sequence. The document outlines the process of PCR including denaturing, annealing and extending DNA strands. It discusses the advantages of PCR being quick, reliable and sensitive for detecting specific pathogens or genes. However, it also notes disadvantages like the need for equipment and risks of contamination producing false results. Examples of PCR applications include detecting viruses, bacteria, and analyzing antibiotic resistance genes.
Enabling RNA-Seq With Limited RNA Using Whole Transcriptome AmplificationQIAGEN
RNA-Seq was developed to perform transcriptome profiling and provides a highly precise measurement of expression levels of transcripts and their isoforms. Normally, RNA-Seq analysis requires at least 500 ng –1 μg of total RNA. When working with small biopsies, single cells (such as circulating tumor cells), or other limited material, whole transcriptome amplification (WTA) is normally required. Various WTA methods overcome limited RNA availability and enable transcriptome analysis from limited material or even single cells. In standard PCR-based WTA procedures, however, bias from uneven coverage of cDNA regions with high GC or AT content or amplification errors can lead to the loss of transcripts and wrong variant calling. Here, we compare a standard RNA-Seq library preparation method and the REPLI-g RNA library protocol. The REPLI-g procedure is a PCR-free protocol to efficiently generate RNA-Seq libraries from small amounts of RNA or a single cell in 6.5–7 hours. The REPLI-g protocol uses whole transcriptome amplification based on multiple displacement amplification (MDA), combined with an efficient library adaptor ligation procedure, to prepare RNA-Seq libraries from small RNA amounts. The procedure demonstrates high fidelity, minimal bias and retention of sample‘s transcriptional profile. Compared to standard RNA-Seq library prep, the REPLI-g protocol demonstrates similar reproducibility and sensitivity in transcript detection.
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.
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
Automated Nucleic Acid Purification from Diverse Sample types using dedicated...QIAGEN
This webinar will focus on the automation of QIAGEN’s new line of DNA and RNA sample prep kits for the microbiome. We will show how automation on the QIAcube enables efficient and reliable use of these samples for sensitive downstream applications such as qPCR and NGS. In addition, you will learn how to successfully use the CLC Microbial Genomics Module for metagenome sequencing and identification of microbial composition and diversity.
This document discusses various nucleic acid amplification tests (NAATs) for detecting Chlamydia trachomatis, including PCR, real-time PCR, transcription-mediated amplification (TMA), and strand displacement amplification (SDA). It provides details on commercially available tests like Cobas Amplicor, Roche cobas, Abbott RealTime, Gen-Probe Aptima Combo 2, and BD ProbeTec systems. The advantages and limitations of each method are outlined. Sensitivity and specificity rates for different sample types are generally high, ranging from 92-100% sensitivity and 95-100% specificity.
Single-nucleotide polymorphisms (SNPs) provide important information about the biology and evolution of different organisms. SNPs may also help predict an individual’s response to certain drugs, susceptibility to environmental factors, and risk of developing particular diseases providing valuable insight into pathophysiology of the human condition. As a result, SNPs with important functional roles often become subjects for high-throughput experiments.
In this webinar, Daniel Tsang provides an overview of genotyping using real-time PCR (qPCR) technology, including challenges and ways to overcome these challenges. He presents a novel qPCR-based genotyping solution, the rhAmp™ SNP Genotyping System, along with its advantages in genotyping, details on cluster separation, as well as solutions to improve the calling accuracy and confidence of making genotype calls.
The document discusses using NCBI databases to design quantitative PCR (qPCR) assays. It describes several NCBI tools that can be used:
1) The NCBI Nucleotide and Gene databases to obtain sequence information for the gene of interest.
2) NCBI BLAST to perform sequence searches and check primer specificity against relevant databases.
3) NCBI dbSNP to search for single nucleotide polymorphisms (SNPs) in the primer binding sites that could affect assay performance.
The document provides guidance on how to use these NCBI tools at various steps of the qPCR assay design process.
QIAcube® RNA isolation from stool samples using the RNeasy® PowerMicrobiome® ...QIAGEN
This application note demonstrates that RNA is extracted efficiently from stool samples using the RNeasy PowerMicrobiome Kit and the QIAcube system. Furthermore, the RNA isolated with the RNeasy PowerMicrobiome Kit and the QIAcube system is compatible with downstream applications.
Semi Automated Low-throughput Workflow for Microbial Analyses of Human StoolQIAGEN
The gut microbiota composition changes dramatically throughout aging and disease. A healthy gut microbiota is typically characterized by large bacterial taxonomic diversity and functional capacity, whereas frailty and aging are associated with loss of diversity and expansion of more pathogenic bacterial species. However, in order to accurately profile changes in microbial communities, the reproducible isolation of high-quality DNA is an important step. Automation enables reliable and reproducible isolation of DNA of superior quality, which can be used directly for downstream sequencing applications.
This webinar focuses on the development of a semi-automated workflow to profile the gut microbiota of young and old individuals and identify changes in bacterial composition and function that occur with age. This workflow will help to simplify and streamline the DNA extraction process for samples with high inhibitor content and subsequent microbial community analyses.
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.
RNA Integrity and Quality – Standardize RNA Quality Control QIAGEN
This document discusses RNA quality control and integrity. It emphasizes that RNA integrity is critical for obtaining accurate gene expression measurements. The RNA Integrity Number (RIN) provides a standardized score to assess RNA integrity based on capillary electrophoresis. Maintaining high RNA purity and avoiding degradation are important to ensure stable RNA samples that can be reliably stored. The QIAxpert system allows comprehensive RNA quality control by assessing concentration, purity, integrity, and contaminants in a single analysis.
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.
Site directed mutagenesis of β2-microglobulin PowerPoint PresentationTyler Liang
This document describes site-directed mutagenesis experiments performed on beta-2 microglobulin (β2m) to study its role in amyloidosis. Primers were designed to introduce mutations D39V and I5T into β2m. Polymerase chain reaction and transformation were used to generate mutant plasmids, which were sequenced to confirm the mutations. Future studies are planned to introduce mutations W61F and W96F to further study β2m stability and aggregation.
Codon bias as a means to fine tune geneAisha Hussain
This document discusses codon bias and how it can be used to fine-tune gene expression. It explains that the genetic code is degenerate, with multiple codons encoding the same amino acid. Organisms exhibit codon bias, preferring certain codons. Codon bias correlates with tRNA abundance and affects translation efficiency. Rare codons can slow translation to regulate co-translational folding and membrane translocation. Codon landscapes within genes also influence expression. Exploiting codon bias through tRNA supplementation or gene optimization improves heterologous protein production. Further integrating experimental data will advance understanding and synthetic biology applications of codon bias.
This document discusses protein engineering through directed evolution. It explains that directed evolution involves randomly recombining genes from protein libraries and screening mutant proteins for improved functions. This leaves beneficial changes up to chance but generates diversity. Rational design is also used to map important protein interactions to guide evolution. The document provides an example of using directed evolution to modify a cytochrome P450 enzyme to more efficiently convert alkanes to alcohols over multiple generations. However, it notes that expressing evolved proteins in vivo and requiring expensive cofactors limit the commercial potential of this approach.
Site-directed mutagenesis is a technique that allows researchers to introduce specific changes to DNA sequences. This allows a single amino acid in a protein to be selectively replaced with another amino acid by modifying the codon. The basic mechanism involves using a primer, polymerase, and replication to introduce a point mutation. Site-directed mutagenesis has applications in protein engineering, studying protein structure and function, and improving traits like enzyme activity or nutrient content.
Protein engineering involves modifying protein structure using recombinant DNA technology or chemical treatment to improve function for use in medicine, industry, and agriculture. The objectives of protein engineering are to create superior enzymes for specific chemical production, produce enzymes in large quantities, and produce superior biological compounds. Protein engineering aims to alter properties like kinetic properties, thermostability, stability in nonaqueous solvents, substrate specificity, and cofactor requirements to meet industrial needs. Common methods for protein engineering include mutagenesis, selection, and recombinant DNA technology.
Site-directed mutagenesis is a technique used to generate specific mutations in DNA at predetermined locations. It involves using a synthetic oligonucleotide primer containing the desired mutation to introduce changes into the DNA sequence during in vitro DNA replication or PCR. This allows researchers to study the effects of mutations and engineer proteins with improved or customized properties. Common methods for site-directed mutagenesis include using single or double primers, cassette mutagenesis by replacing DNA fragments, and PCR-based mutagenesis. The technique has various applications in investigating protein function and developing proteins for commercial uses.
Artificial vectors such as bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), P1-derived artificial chromosomes (PACs), and human artificial chromosomes (HACs) can carry large DNA fragments for research purposes. BACs can hold up to 300kb of DNA and are used to sequence genomes. YACs can carry 500kb of DNA but are prone to rearrangement. HACs are separate microchromosomes that act as new chromosomes and can carry therapeutic genes for research including disease models.
The document describes Cignal Lenti Reporter Assays which use lentiviral particles containing pathway-specific reporter constructs to enable signaling studies in difficult to transfect cell types. Key points:
1) Cignal Lenti Reporters contain optimized transcriptional response elements and reporter genes for high performance and sensitivity in signaling studies across 45 pathways.
2) The assays allow transient transduction studies or generation of stable pathway sensor cell lines for both easy and difficult to transfect cell types like primary cells and stem cells.
3) The lentiviral particles are ready to use with no need for viral generation or titering, and the assays provide a complete solution for signaling studies.
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.
Cell-based Reporter Assays: Measure 45 Signaling Pathway Activity in Any Cel...Qiagen - Egypt
Would you like to measure signaling pathway activity in your favorite cell? Learn how to successfully apply convenient and robust reporter assays to your RNA interference, gene over-expression, protein, or small molecule studies. The Cignal Reporter Assays are an excellent tool for studying pathway signaling activity in cells that are amenable to transfection, available for studying numerous pathways including (ROS, Wnt, NF-kB, Notch, cAMP/PKA, TGFbeta, and the Cignal Lenti Reporter Assays combines the power of a lentiviral delivery system with our robust transcription factor reporter technology, enabling you to study signal pathways in virtually any cell type. You can find a technology overview, protocol tutorial, and application examples in the following presentation.
Cell-based Reporter Assays: Measure 45 Signaling Pathway Activity in Any Cell...QIAGEN
Would you like to measure signaling pathway activity in your favorite cell? Learn how to successfully apply convenient and robust reporter assays to your RNA interference, gene over-expression, protein, or small molecule studies. The Cignal Reporter Assays are an excellent tool for studying pathway signaling activity in cells that are amenable to transfection, available for studying numerous pathways including (ROS, Wnt, NF-kB, Notch, cAMP/PKA, TGFbeta, and the Cignal Lenti Reporter Assays combines the power of a lentiviral delivery system with our robust transcription factor reporter technology, enabling you to study signal pathways in virtually any cell type. You can find a technology overview, protocol tutorial, and application examples in the following presentation.
1. The GeneXpert system uses real-time PCR and molecular beacons to detect Mycobacterium tuberculosis (MTB) and rifampicin resistance directly from sputum samples.
2. The Xpert MTB/RIF test was improved with the Xpert Ultra, which features increased sensitivity, the ability to detect additional MTB genes, and modified molecular beacons to better identify rifampicin resistance mutations using melting curve analysis.
3. Studies showed the Xpert Ultra had a lower limit of detection of 16 CFU/ml compared to 131 CFU/ml for Xpert MTB/RIF, and was better able to detect heteroresistance and mixed infections
QIAseq Targeted DNA, RNA and Fusion Gene PanelsQIAGEN
Tumor heterogeneity has been known for a while but quantifying heterogeneity is still a challenge. NGS is the method of choice in the analysis of tumor heterogeneity, however, there are some inherent challenges associated with it. These include false positives, gaps in the gene due to overrepresentation and incomplete representation of low-frequency transcripts – all contributing to an inaccurate picture. Conventional library prep strategies for NGS are based on PCR, which introduces sequence-based bias and amplification noise, leading to these inaccuracies. In this webinar, we will cover
1. Principles of UMI and the new QIAseq product porfolio
2. How UMI along with SPE (single primer extension) allows for increased uniformity across difficult-to-sequence regions, removal of library construction bias, improved data analysis and sequencing optimization
3. How data generated from using UMI and SPE is directly comparable to analysis derived from whole transcriptome and exome sequencing
4. Application of UMI and SPE in the discovery of novel gene fusions and in the analysis of gene expression and genetic variation
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
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.
This document discusses wet-lab considerations for Illumina sequencing data analysis. It describes the typical Illumina sequencing workflow including library preparation, cluster formation, sequencing, and data analysis. It provides details on DNA and RNA input requirements, library construction steps like fragmentation and adapter ligation, and quality control methods. The document also discusses newer sequencing technologies like Pacific Biosciences and Oxford Nanopore sequencing.
The document discusses RNA interference (RNAi) solutions for gene knockdown research. It describes using short interfering RNA (siRNA) and short hairpin RNA (shRNA) to temporarily or permanently suppress gene expression. Challenges of RNAi experiments include off-target effects and variability in knockdown efficiency. The document promotes solutions from QIAGEN, including validated siRNA and shRNA designs with controls, to help optimize RNAi experiments and reduce non-specific effects.
This document outlines the workflow and methods for an RNA lab involving isolation of RNA from Arabidopsis leaves, quantification of the RNA, reverse transcription to cDNA, and quantitative PCR (qPCR) analysis. The workflow takes place over two days, with RNA isolation and quantification on day 1 and setting up and analyzing qPCR on day 2. Students will be evaluated through an in-class quiz worth 50 points and a lab report worth 50 points. The document provides details on the RNA isolation, quantification and quality check methods, as well as background information and considerations for the qPCR analysis.
This document provides an overview of RT2 Profiler PCR Arrays from SABiosciences, which allow for gene expression analysis from small samples and FFPE samples. The document discusses how PCR Arrays work using SABiosciences' PreAMP technology to increase sensitivity for samples containing as little as 1-100ng of RNA. It also reviews the performance data demonstrating the ability of PreAMP to detect more genes and shift genes with high Ct values into the detectable range. Finally, it highlights the complete PCR Array system from SABiosciences which provides optimized kits, controls, and software for reliable gene expression analysis from sample to results in about 3 hours.
Please note: This presentation accompanies a recorded webinar at:
https://www1.gotomeeting.com/register/347794241
Biomarkers for studying gene regulation and cell function can be efficiently analyzed by multiplexed methods. Dr. Jim Lazar from OriGene Technologies will provide an overview of four different but related detection technologies that can be used to analyze genetic variants, microRNA expression, transcription factor binding, and protein expression on the Luminex xMAP platform. OriGene’s broad panel of assays and tools for discovery, analysis and validation of multiple classes of important biomarkers will allow researcher to develop more accurate descriptions of biologically complex systems.
Genomics research and discovery has led to a large increase of reported single nucleotide polymorphisms (SNPs). From 2006 to 2017, the number of refSNPs in the NCBI dbSNP database has increased 13-fold. Many polymorphisms can be linked to disease susceptibility and responses to chemical therapies. Other polymorphisms are used as trait identifiers in livestock and plants. Being able to inexpensively and accurately determine the genotype in high-throughput fashion, with low sample input is a critical need in current, large-scale screening efforts. In this presentation, we present a novel, probe-based, PCR genotyping solution that possesses the universal cycling conditions, strong signal generation, and benchtop reaction stability needed for high-throughput screening. We also present the mechanism and unique technical advantages of using the rhAmp SNP Genotyping System, and we will illustrate how easy it is to generate high quality genotyping data.
This document describes 10 different types of PCR: 1) Conventional DNA based PCR, 2) Reverse transcription-PCR, 3) Asymmetric PCR, 4) Inverse PCR, 5) Nested PCR, 6) Anchored PCR, 7) PCR using other polymerases, 8) In situ PCR, 9) Real-Time PCR, and 10) Multiplex PCR. It provides details on the methodology and applications of each type of PCR.
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.
Introduction to Real Time PCR (Q-PCR/qPCR/qrt-PCR): qPCR Technology Webinar S...QIAGEN
This slidedeck introduces the concepts of real-time PCR and how to conduct a real-time PCR assay. The topics that are covered include an overview of real-time PCR chemistries, protocols, quantification methods, real-time PCR applications and factors for success.
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.
Similar to Cignal reporter assays webinar 2013 (20)
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. Cignal™ Reporter Assays
Functional Analysis of Genes
Biologics and Small Molecule Compounds
Krishnan Allampallam, Ph.D.,MBA
PCR Array & Pathway Signaling
The Cignal Reporter Assays are intended for molecular biology applications. This product is
not intended for the diagnosis, prevention or treatment of a disease.
For Internal Use Only
-1-
Sample & Assay Technologies
2. Topics to be covered
■ Introduction
■ What are Cignal Reporter Assays
■ How Cignal Reporter Assays work
■ How you can use Cignal Reporter
Assays in your research
For Internal Use Only
-2-
Sample & Assay Technologies
6. Cell-based assays
Research Challenges
■ Intra-experiment variability
Well-to-well, plate-to-plate reproducibility
■ Inter-experiment variability
“status” of the cells
who in the lab is conducting the experiments.
■ Assay performance
Sensitivity, specificity, signal to noise ratio, and simplicity
For Internal Use Only
-6-
Sample & Assay Technologies
7. Cignal™ Reporter Assays
Flexible, pre-optimized solution for cell based assays
■ Functionally validated dual-reporter formulation (Luciferase)
Minimizes experimental variability, increasing biological relevance
■ Ready-to-use reporters and controls, in transfection ready format
Enables simple, rapid analysis of the regulation of 45 signal transduction pathways
■ Genetically engineered transcriptional regulatory elements (TREs) &
Destabilized , codon –optimized firefly luciferase
Increases the signal to noise ratio, maximizes assay sensitivity and specificity
Available in both Luciferase and GFP formats
For Internal Use Only
-7-
Sample & Assay Technologies
8. Topics to be covered
■ Introduction
■ What are Cignal Reporter Assays
Challenges associated
Solution: Cignal reporter Assay
■ How Cignal Reporter Assays work
Complete Product Portfolio
Dual-Luciferase Assays
High performace Luciferase
Pre-optimized TREs
■ How you can use Cignal Reporter
Assays in your research
For Internal Use Only
-8-
Sample & Assay Technologies
9. Cignal™ Reporter AssaysComplete Solution
Cignal Reporter Assays:
Flexible solution: Multiple Reporter Formats
Pathway-targeted Transcriptional Regulatory Elements
(TRE), which establish the
pathway specificity of each reporter
TATA
box
Reporter Construct
GFP/firefly luciferase
Tandem repeats of
TRE
For Internal Use Only
-9-
Reporter Protein is
expressed when TF
binds to TRE
Sample & Assay Technologies
10. Cignal™ Reporter Assays
Flexible solution: Multiple Reporter Formats
Pathway-targeted Transcriptional Regulatory Elements
(TRE), which establish the
pathway specificity of each reporter
TATA
box
Reporter Construct
GFP/firefly luciferase
Tandem repeats of
TRE
EGFP
TF
FL
Upstream Signaling
Events
For Internal Use Only
- 10 -
Sample & Assay Technologies
11. Cignal™ Reporter Assays
Multiple Reporter Formats – Complete dual luciferase system
Pathway-targeted Transcriptional Regulatory Elements
(TRE), which establish the
pathway specificity of each reporter
TATA
box
Reporter Construct
firefly luciferase
Tandem repeats of
TRE
TATA
box
Negative Control Construct
firefly luciferase
firefly luciferase
Positive Control Construct
CMV
TATA
box
Internal Control Construct
CMV
TATA
box
For Internal Use Only
- 11 -
Renilla Luciferase
Sample & Assay Technologies
12. Cignal™ Reporter Assays
Plasmid - 45 Pathways
Pathway
Amino Acid Deprivation
Androgen
Antioxidant Response
ATF6
C/EBP
cAMP/PKA
Cell Cycle
DNA Damage
EGR1
ER Stress
Estrogen
GATA
Glucocorticoid
Heat Shock Response
Heavy Metal Stress
Hedgehog
HNF4
Hypoxia
Interferon Regulation
Type I Interferon
Interferon Gamma
KLF4
Liver X
For Internal Use Only
Transcription Factor
ATF4/ATF3/ATF2
Androgen Receptor
Nrf2 & Nrf1
ATF6
C/EBP
CREB
E2F/DP1
p53
EGR1
CBF/NF-Y/YY1
ER
GATA
GR
HSF
MTF1
Gli
HNF4
HIF-1
IRF-1
STAT1/STAT2
STAT1/STAT1
KLF4
LXR
- 12 -
Pathway
MAPK/ERK
MAPK/JNK
MEF2
c-myc
Nanog
NFκB
Notch
Oct4
Pax6
PI3K/AKT
PKC/Ca++
PPAR
Progesterone
Retinoic Acid
Retinoid X
Sox2
SP1
STAT3
TGFβ
VDRE
Wnt
Xenobiotic
Transcription Factor
Elk-1/SRF
AP-1
MEF2
Myc/Max
Nanog
NFκB
RBP-Jk
Oct4
Pax6
FOXO
NFAT
PPAR
PR
RAR
RXR
Sox2
SP1
STAT3
SMAD2/3/4
Vitamin D Receptor
TCF/LEF
AhR
Sample & Assay Technologies
13. Cignal™ Reporter Assays
Product offering
Primary cells, stem cells,
and difficult to transfect cell lines
Easy to
transfect cell lines
Endpoint
Assays
Cignal Dual-Luciferase
Reporter Assays
Single Pathway
Assays
10-Pathway
Arrays
Dynamic
Live Cell Assays
Cignal GFP
Reporter Assays
For Internal Use Only
Cignal Lenti
Luciferase Reporters
Single Pathway
Assays
Multiple options to suit
your research needs:
Dynamic
Live Cell Assays
Endpoint
Assays
Single Pathway
Assays
10-Pathway
Arrays
Cignal Lenti
GFP Reporters
Single Pathway
Assays
Plasmid and Lentiviral, Luciferase and GFP reporters,
Single, 10-pathway, 45-pathway
- 13 -
Sample & Assay Technologies
14. Cignal™ Reporter Assays
Lentiviral Assays (35 Pathways)
Pathway
Amino Acid Deprivation
Androgen
Antioxidant Response
ATF6
C/EBP
cAMP/PKA
Cell Cycle
DNA Damage
EGR1
ER Stress
Estrogen
GATA
Glucocorticoid
Heat Shock Response
Heavy Metal Stress
Hedgehog
HNF4
Hypoxia
Interferon Regulation
Type I Interferon
Interferon Gamma
KLF4
Liver X
For Internal Use Only
Transcription Factor
ATF4/ATF3/ATF2
Androgen Receptor
Nrf2 & Nrf1
ATF6
C/EBP
CREB
E2F/DP1
p53
EGR1
CBF/NF-Y/YY1
ER
GATA
GR
HSF
MTF1
Gli
HNF4
HIF-1
IRF-1
STAT1/STAT2
STAT1/STAT1
KLF4
LXR
- 14 -
Pathway
MAPK/ERK
MAPK/JNK
MEF2
c-myc
Nanog
NFκB
Notch
Oct4
Pax6
PI3K/AKT
PKC/Ca++
PPAR
Progesterone
Retinoic Acid
Retinoid X
Sox2
SP1
STAT3
TGFβ
VDRE
Wnt
Xenobiotic
Transcription Factor
Elk-1/SRF
AP-1
MEF2
Myc/Max
Nanog
NFκB
RBP-Jk
Oct4
Pax6
FOXO
NFAT
PPAR
PR
RAR
RXR
Sox2
SP1
STAT3
SMAD2/3/4
Vitamin D Receptor
TCF/LEF
AhR
Sample & Assay Technologies
15. Using Cignal™ Reporter Assays
What you need
• Cignal Reporter Assay
• Reliable transfection reagent and
protocol (Attractene, HiPerFect)
• Test Biological
•shRNA (SureSilencing shRNA)
• expression vectors (QIAgenes)
• protein/peptide
• small molecule
• Detection Instrument and
Luciferase Substrates
For Internal Use Only
- 15 -
Sample & Assay Technologies
16. Cignal™ Reporter Assays
Key advantages – Assay design- Dual Luciferase
Pathway-targeted transcriptional
regulatory elements (45)
Reporter Construct
TRE
TATA
box
Firefly luciferase
Normalization Construct
CMV
TATA
box
Renilla luciferase
Constitutive transcriptional
regulatory element (1)
The Transcriptional Regulatory Elements (TREs) establishes the
pathway specificity of each reporter!
For Internal Use Only
- 16 -
Sample & Assay Technologies
17. Cignal™ Reporter Assays
Why Dual Luciferase
Sources of Variability in Cell-based Assays:
• Number of cells seeded per well
• Transfection efficiencies within and across plates
• Multichannel pipettor inconsistencies
• “Edge effects” influencing cell culture
• Viability of cells following transfection/treatment
• Lysis efficiencies within and across plates
• Variability inherent to reporter assay
Dual-Luciferase Assay
design corrects for
these unwanted
sources of variability.
• Changes due to the designed treatments of cells
• RNA interference
• Overexpression
• Recombinant protein/peptide/growth factor
• Small molecule
For Internal Use Only
- 17 -
Sample & Assay Technologies
24. Cignal™ Reporter Assays
Complete GFP system: to study interactions in individual cells
Pathway-targeted Transcriptional Regulatory Elements
(TRE), which establish the
pathway specificity of each reporter
TATA
box
GFP
TATA
box
Reporter Construct
GFP
TATA
box
GFP
Tandem repeats of
TRE
Negative Control Construct
Positive Control Construct
For Internal Use Only
CMV
- 24 -
Sample & Assay Technologies
25. Cignal™ Reporter Assays
DNA-Vector GFP – 16 pathways
Pathway
Amino Acid Deprivation
Androgen
Antioxidant Response
ATF6
C/EBP
cAMP/PKA
Cell Cycle
DNA Damage
EGR1
ER Stress
Estrogen
GATA
Glucocorticoid
Heat Shock Response
Heavy Metal Stress
Hedgehog
HNF4
Hypoxia
Interferon Regulation
Type I Interferon
Interferon Gamma
KLF4
Liver X
For Internal Use Only
Transcription Factor
ATF4/ATF3/ATF2
Androgen Receptor
Nrf2 & Nrf1
ATF6
C/EBP
CREB
E2F/DP1
p53
EGR1
CBF/NF-Y/YY1
ER
GATA
GR
HSF
MTF1
Gli
HNF4
HIF-1
IRF-1
STAT1/STAT2
STAT1/STAT1
KLF4
LXR
- 25 -
Pathway
MAPK/ERK
MAPK/JNK
MEF2
c-myc
Nanog
NFκB
Notch
Oct4
Pax6
PI3K/AKT
PKC/Ca++
PPAR
Progesterone
Retinoic Acid
Retinoid X
Sox2
SP1
STAT3
TGFβ
VDRE
Wnt
Xenobiotic
Transcription Factor
Elk-1/SRF
AP-1
MEF2
Myc/Max
Nanog
NFκB
RBP-Jk
Oct4
Pax6
FOXO
NFAT
PPAR
PR
RAR
RXR
Sox2
SP1
STAT3
SMAD2/3/4
Vitamin D Receptor
TCF/LEF
AhR
Sample & Assay Technologies
26. Cignal™ Reporter Assays
Lenti- GFP Reporter – 6 pathways
Pathway
Amino Acid Deprivation
Androgen
Antioxidant Response
ATF6
C/EBP
cAMP/PKA
Cell Cycle
DNA Damage
EGR1
ER Stress
Estrogen
GATA
Glucocorticoid
Heat Shock Response
Heavy Metal Stress
Hedgehog
HNF4
Hypoxia
Interferon Regulation
Type I Interferon
Interferon Gamma
KLF4
Liver X
For Internal Use Only
Transcription Factor
ATF4/ATF3/ATF2
Androgen Receptor
Nrf2 & Nrf1
ATF6
C/EBP
CREB
E2F/DP1
p53
EGR1
CBF/NF-Y/YY1
ER
GATA
GR
HSF
MTF1
Gli
HNF4
HIF-1
IRF-1
STAT1/STAT2
STAT1/STAT1
KLF4
LXR
- 26 -
Pathway
MAPK/ERK
MAPK/JNK
MEF2
c-myc
Nanog
NFκB
Notch
Oct4
Pax6
PI3K/AKT
PKC/Ca++
PPAR
Progesterone
Retinoic Acid
Retinoid X
Sox2
SP1
STAT3
TGFβ
VDRE
Wnt
Xenobiotic
Transcription Factor
Elk-1/SRF
AP-1
MEF2
Myc/Max
Nanog
NFκB
RBP-Jk
Oct4
Pax6
FOXO
NFAT
PPAR
PR
RAR
RXR
Sox2
SP1
STAT3
SMAD2/3/4
Vitamin D Receptor
TCF/LEF
AhR
Sample & Assay Technologies
27. Cignal™ Reporter Assays
GFP system application: Small molecule studies
Fluorometry
Fluorescent Microscopy
Treatment of Cignal SRE (GFP) transfectants with 10% serum and 10 ng/ml PMA
activates the MAPK/ERK signaling pathway.
For Internal Use Only
- 27 -
Sample & Assay Technologies
28. Topics to be covered
■ Introduction to SABiosciences
■ What are Cignal Reporter Assays
Challenges associated
Solution: Cignal reporter Assay
■ How Cignal Reporter Assays work
Complete Product Portfolio
Dual-Luciferase Assays
High performace Luciferase
Pre-optimized TREs
■ How you can use Cignal Reporter
Assays in your research
For Internal Use Only
- 28 -
Sample & Assay Technologies
29. Cignal™ Reporter Assays
Application in multiple research areas
Functional Genomics
What’s the phenotype of my gene?
Does my gene of interest regulate signaling pathway “X”?
Functional Proteomics
What’s the phenotype of my protein/peptide?
Does my protein, peptide, or growth factor of interest regulate signaling
pathway “X”?
Drug Screening
What is the mechanism of action of my small molecule drug candidates?
Do my drug candidates of interest regulate signaling pathway “X”?
For Internal Use Only
- 29 -
Sample & Assay Technologies
31. Cignal™ Reporter Assays
Application in multiple research areas
Functional Genomics
What’s the phenotype of my gene?
Does my gene of interest regulate signaling pathway “X”?
Functional Proteomics
What’s the phenotype of my protein/peptide?
Does my protein, peptide, or growth factor of interest regulate signaling
pathway “X”?
Drug Screening
What is the mechanism of action of my small molecule drug candidates?
Do my drug candidates of interest regulate signaling pathway “X”?
For Internal Use Only
- 31 -
Sample & Assay Technologies
33. Cignal™ Reporter Assays
Application in multiple research areas
Functional Genomics
What’s the phenotype of my gene?
Does my gene of interest regulate signaling pathway “X”?
Functional Proteomics
What’s the phenotype of my protein/peptide?
Does my protein, peptide, or growth factor of interest regulate signaling
pathway “X”?
Drug Screening
What is the mechanism of action of my small molecule drug candidates?
Do my drug candidates of interest regulate signaling pathway “X”?
For Internal Use Only
- 33 -
Sample & Assay Technologies
35. Cignal reporter assays
Research challenge
Application – Study pathway interaction
Pathway Interaction Study
Situation
I have a biological molecule that knocks down p53 expression
Question
1. What is impact of p53 gene expression knock down on
the p53 signaling pathway?
2. What is the impact of p53 knock down on other cancerrelevant signaling pathways?
For Internal Use Only
- 35 -
Sample & Assay Technologies
37. Cignal™ Reporter Assays
Application – study pathway interaction
Approach:
Carry out a p53 RNA interference experiment with
multiple cancer-relevant Cignal Reporter Assays
For Internal Use Only
- 37 -
Sample & Assay Technologies
38. Cignal Finder arrays
Cancer array design
Pathway
Cell Cycle
DNA Damage
Hypoxia
MAPK/ERK
MAPK/JNK
c-Myc
NFkB
Notch
TGFβ
Wnt
For Internal Use Only
Transcription Factor
E2F/DP1
p53
HIF
Elk-1/SRF
AP1
Myc/Max
NFkB
RBP-Jk
SMAD2/SMAD3/SMAD4
TCF/LEF
- 38 -
Sample & Assay Technologies
39. Cignal Finder array
How does it work?
For Internal Use Only
- 39 -
Sample & Assay Technologies
40. Cignal Finder cancer array
p53 RNAi study design
1.
p53 Cignal Reporter
2.
RBP-Jk Cignal Reporter
3.
TCF/LEF Cignal Reporter
4.
E2F Cignal Reporter
5.
AP1 Cignal Reporter
6.
SRE Cignal Reporter
7.
SMAD Cignal Reporter
8.
NFkB Cignal Reporter
9.
Myc Cignal Reporter
1
2
4
3
5
6
7
8
9 10 11 12
p53 siRNA
Neg. control siRNA
10. HIF Cignal Reporter
11. Cignal Negative Control
12. Cignal Positive Control
For Internal Use Only
- 40 -
Sample & Assay Technologies
41. Cignal Finder cancer array
p53 RNAi study design - results
Impact of p53 siRNA Treatment
For Internal Use Only
- 41 -
Sample & Assay Technologies
42. Cignal Finder cancer array
p53 RNAi study design – next steps
p53 Signaling
Hypoxia
Signaling
MAPK/ERK
Signaling
Notch Signaling
For Internal Use Only
- 42 -
Sample & Assay Technologies
43. Cignal™ Reporter Assays System
Summary
■ Breadth of Pathways and Research Application
■ 45 Pathway-Focused Reporter Assays
■ 6 Application-Specific Cignal Finder 10-Pathway Arrays
■ 1 45-Pathway Reporter Array
■ Performance
■ Exceptional Sensitivity, Specificity, Signal-to-Noise Ratio, and
Reproducibility
■ Convenience
■ Ready-to-use, validated, preformatted transient assays
■ Dual Modalities
■ Dual-luciferase quantitative endpoint assays
■ GFP dynamic live cell assays with single cell resolution
For Internal Use Only
- 43 -
Sample & Assay Technologies
44. New research tool for 2013
http://www.sabiosciences.com/sciglobe.php
Sample & Assay Technologies
45. Cignal™ Reporter Assays
For rapidly analyzing pathway signaling activity
Questions?
Contact Technical Support 9 AM – 6 PM Eastern M – F
Telephone: 888-503-3187
Email: support@SABiosciences.com
Thank you!
Krishnan Allampallam
QIAwebinars@qiagen.com
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
For Internal Use Only
- 45 -
Sample & Assay Technologies