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 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.
RT2 Profiler PCR Arrays are a real-time PCR technology that allows researchers to study gene expression patterns across biological pathways and processes. The arrays contain pre-designed primer assays for 84 relevant genes as well as controls on a single plate in a 96-well format. The gene content of the arrays is selected based on biological relevance and published associations with relevant pathways. The primer assays on the arrays undergo extensive validation for sensitivity, specificity, reproducibility, and amplification efficiency. The PCR Array system also includes optimized components for RNA isolation, cDNA synthesis, and real-time PCR to provide a complete validated workflow for gene expression analysis from sample to results.
The ChIP-qPCR assays provide pre-designed and validated real-time PCR primer assays that measure genomic DNA enrichment within chromatin immunoprecipitation samples. They save researchers time and money by eliminating the need to design their own assays. The assays provide quick, easy, and quantitative analysis of multiple promoter regions from a single ChIP sample using real-time PCR, addressing challenges of traditional ChIP workflows. The assays offer comprehensive coverage of human, mouse, and rat genomes and can be customized, allowing researchers to expand their ChIP experiments.
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.
The SlipChip is a simple lab-on-a-chip system that can perform many reactions in parallel in a small volume. It is used to demonstrate digital PCR, which can quantify DNA over a large concentration range by loading samples into wells and counting the number of wells that test positive. The SlipChip allows running multiple digital PCR experiments simultaneously on different sections of the chip, improving quantification accuracy and dynamic range. Examples show detecting pathogens in blood and simultaneously measuring viral loads of HCV and HIV. The system is flexible and its applications are limited only by the assays designed for it. Feedback is sought on potential clinical and research uses.
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!
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.
This document describes PrimeTime® qPCR products for gene expression analysis, including probe- and primer-based assays for human, mouse, and rat sequences as well as custom assays. It provides details on master mixes, probes, controls, and an assay design and ordering process to ensure specific and efficient assays. The assays are guaranteed to have high efficiency and sensitivity for accurate quantification of gene expression.
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.
RT2 Profiler PCR Arrays are a real-time PCR technology that allows researchers to study gene expression patterns across biological pathways and processes. The arrays contain pre-designed primer assays for 84 relevant genes as well as controls on a single plate in a 96-well format. The gene content of the arrays is selected based on biological relevance and published associations with relevant pathways. The primer assays on the arrays undergo extensive validation for sensitivity, specificity, reproducibility, and amplification efficiency. The PCR Array system also includes optimized components for RNA isolation, cDNA synthesis, and real-time PCR to provide a complete validated workflow for gene expression analysis from sample to results.
The ChIP-qPCR assays provide pre-designed and validated real-time PCR primer assays that measure genomic DNA enrichment within chromatin immunoprecipitation samples. They save researchers time and money by eliminating the need to design their own assays. The assays provide quick, easy, and quantitative analysis of multiple promoter regions from a single ChIP sample using real-time PCR, addressing challenges of traditional ChIP workflows. The assays offer comprehensive coverage of human, mouse, and rat genomes and can be customized, allowing researchers to expand their ChIP experiments.
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.
The SlipChip is a simple lab-on-a-chip system that can perform many reactions in parallel in a small volume. It is used to demonstrate digital PCR, which can quantify DNA over a large concentration range by loading samples into wells and counting the number of wells that test positive. The SlipChip allows running multiple digital PCR experiments simultaneously on different sections of the chip, improving quantification accuracy and dynamic range. Examples show detecting pathogens in blood and simultaneously measuring viral loads of HCV and HIV. The system is flexible and its applications are limited only by the assays designed for it. Feedback is sought on potential clinical and research uses.
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!
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.
This document describes PrimeTime® qPCR products for gene expression analysis, including probe- and primer-based assays for human, mouse, and rat sequences as well as custom assays. It provides details on master mixes, probes, controls, and an assay design and ordering process to ensure specific and efficient assays. The assays are guaranteed to have high efficiency and sensitivity for accurate quantification of gene expression.
This document discusses using real-time PCR and end-point PCR for gene expression analysis and microarray data verification. Real-time PCR provides a very quantitative and high sensitivity method for determining gene expression levels across a wide dynamic range but requires specialized equipment. End-point PCR provides a less quantitative but still useful method when used with an internal normalizer to control for systematic variation, and does not require specialized equipment. Both methods are useful for direct expression profiling and verification of microarray results on a gene-by-gene basis using commercially available assay kits.
Real-Time quantitative PCR (qPCR) is a mainstream method that is used in research and diagnostic applications for quantification of gene expression. IDT has developed a robust and affordable qPCR master mix for use with probe-based qPCR in single and multiplex assays. In this presentation, we explore a variety of applications of PrimeTime® Gene Expression Master Mix. We cover the use of PrimeTime master mix with probe based assays from IDT. We also look at the use of PrimeTime master mix in multiplex applications without the loss of sensitivity that is commonly observed. Finally, we demonstrate the unmatched stability of PrimeTime master mix under ambient temperatures, saving your research money and minimizing on shipping delays.
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.
Critical Factors for Successful Real-Time PCR: Multiplex PCRQIAGEN
Multiplex end-point PCR is a powerful tool for genotyping and many other applications. QIAGEN’s multiplex PCR chemistry is optimized for reliable amplification of many different templates with high variability in copy numbers. Thus it enables very quick establishment of a new lab routine and instant success for your multiplex PCR strategy.
There is a set of critical factors which we recommend to be regarded for planning and performing this kind of PCR. These will be discussed in detail in the webinar. Additionally, our multiplex PCR chemistry has recently been gaining increasing popularity among scientists who are utilizing it for their next-generation sequencing workflows.
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.
Take your RNA research to the next level with QIAGEN LNA tools!QIAGEN
Download the flyer!
Experience truly exceptional RNA research with QIAGEN's next-generation, LNA®-enhanced tools. LNA (Locked Nucleic Acid) oligos bind with much higher affinity and specificity to RNA targets than standard DNA and RNA oligos – This enables specific and sensitive detection of small RNAs and discrimination between highly similar
sequences.
This document summarizes a presentation about RNase H2 PCR (rhPCR), a new molecular technology that uses an RNA residue in PCR primers and a thermostable RNase H2 enzyme. It describes how rhPCR works, the advantages it provides over traditional PCR including reduced primer-dimer formation and improved specificity for rare allele detection. Two generations of cleavable primer designs - GEN1 and GEN2 - are discussed, along with their different applications. Examples are provided that demonstrate how rhPCR can improve assays for SNP genotyping, multiplex PCR, and detection in complex backgrounds.
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.
Use of CRISPR-Cas9 has revolutionized targeted genome editing. However, rapid design of high-quality guide RNA (gRNA) sequences with high on-target and low off-target editing remains challenging. We implemented a machine learning algorithm to design high-quality gRNA sequences in 5 commonly used species (human, mouse, rat, zebrafish, and nematode). Our tool also designs gRNA sequences against custom targets, and can check existing gRNA designs for quality. In this webinar, we review our data illustrating this tool's performance and demonstrate its use in predicting and designing improved gRNAs for genome editing.
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.
This document provides information about qBiomarker somatic mutation PCR arrays for cancer mutation analysis. It discusses how the arrays can detect mutations in key cancer genes and pathways. The document outlines the workflow, which involves using quantitative PCR to detect mutations in DNA samples. Data is analyzed by comparing Ct values between mutation assays and controls to identify which samples contain mutations. The document provides examples of this analysis method using lung and colon cancer cell line and patient samples.
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 importance of controls and novel solutions for successful real-time qPCRQIAGEN
The increasing demand for streamlined, monitored and ultrafast qPCR procedures requires high-performance, real-time quantitative RT and PCR chemistries. Particularly, procedures utilizing generic kits for gene expression analysis should include in-process safety measures to avoid variables and control accuracy of procedures and results. This slidedeck presents innovative solutions for one-step and two-step RT-PCR that significantly enhance performance and reliability in qRT-PCR. The new QuantiNova kit family offers a combination of various integrated safety features to remove variables and prevent artifacts. Internal control RNA, removal of genomic DNA, room temperature set-up capability for RT-PCR and a built-in visual pipetting control verify accurate procedures, ensuring reliable gene expression profiling.
This slidedeck explains the principles of the technologies and shows data demonstrating performance in qRT-PCR. Find out how you can verify accurate performance in qRT-PCR and improve your results!
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.
Cancer therapies that target specific pathways can be more effective than established, nonspecific chemotherapy and radiation treatments, and may prevent side effects on healthy tissues. Such targeted therapies can only be applied after underlying gene mutations have been identified. However, detecting low frequency variants from clinically relevant samples poses significant challenges. Specimens are routinely formalin-fixed and paraffin-embedded (FFPE) for histology, which can decrease the efficiency of NGS library preparation. In this presentation, we discuss approaches for extraction of DNA from FFPE samples, and recommend quality control assays to guide parameter selection for library construction and sequencing depth.
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.
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
Alzheimer’s disease (AD) is a devastating neurodegenerative disease that is genetically complex. Although great progress has been made in identifying fully penetrant mutations in genes that cause early-onset AD, these still represent a very small percentage of AD cases. Large-scale, genome-wide association studies (GWAS) have identified at least 20 additional genetic risk loci for the more common form: late-onset AD. However, the identified SNPs are typically not the actual risk variants, but are in linkage disequilibrium with the presumed causative variants [1].
To help identify causative genetic variants, we have combined highly accurate, long-read sequencing with hybrid-capture technology. In this collaborative webinar*, we present this method and show how combining IDT xGen® Lockdown® Probes with PacBio SMRT® Sequencing allows targeting and sequencing of candidate genes from genomic DNA and corresponding transcripts from cDNA. Using a panel of target capture probes for 35 AD candidate genes, we demonstrate the power of this approach by looking at data for two individuals with AD. Some additional benefits of this method include the ability to leverage long reads, phase heterozygous variants, and link corresponding transcript isoforms to their respective alleles.
Reference: 1. Van Cauwenberghe C, Van Broeckhoven C, Sleegers K. (2016) The genetic landscape of Alzheimer disease: clinical implications and perspectives. Genet Med, 18(5):421–430.
* This presentation represents a collaboration between Pacific Biosciences and Integrated DNA Technologies. The individual opinions expressed may not reflect shared opinions of Pacific Biosciences and Integrated DNA Technologies.
The CRISPR-Cas9 system has emerged as one of the leading tools for modifying genomes of organisms ranging from E. coli to humans. One of the key components of this editing system is Cas9 endonuclease. The cleavage activity of the S. pyogenes Cas9 enzyme is mediated by the coordinated functions of two catalytic domains and creates blunt-ended, double-stranded breaks. Alanine substitution at key residues within these domains creates two Cas9 nickase variants. Variant D10A produces a nick on the targeting strand, while H840A nicks the non-targeting strand. This double nicking strategy can be leveraged to reduce unwanted off-target effect. However, the nickase experiments can be inherently more complicated than standard CRISPR-Cas9 editing, given the requirement for two guide RNAs to function simultaneously.
In this webinar, both Shuqi Yan and Mollie Schubert present the data from the characterization of a number of factors that impact the efficiency of cooperative nicking in cell cultures. They also summarize a few key design considerations for achieving efficient gene disruption or homology directed repair (HDR) when planning your nickase experiments.
Learn more: http://www.idtdna.com/pages/products/crispr-genome-editing
QIAGEN's qPCR data analysis web portal allows users to analyze quantitative PCR (qPCR) data online. Users can upload raw CT values from SYBR Green or probe-based qPCR assays. The portal will normalize the data, calculate fold changes, and generate graphs and exportable results within 15 minutes. The portal guides users through a seven step process to choose their experiment, upload an Excel file, set up the analysis, view processed results tables and graphs, create additional visualization plots, and download the analyzed data for further exploration. Key benefits include reliable conversion of CT values into fold changes with confidence intervals and p-values, automatic housekeeping gene selection, and suggestions for follow-up experiments.
This study examined the effects of bone marrow cell treatment on salivary glands in mice with Sjögren's syndrome. Researchers extracted mRNA from salivary gland cells of mice with Sjögren's syndrome and used real-time PCR to analyze gene expression. They found that bone marrow cell treatment upregulated genes related to saliva production and downregulated genes associated with inflammation. Specifically, amylase and parotid secretary protein genes were upregulated while the Sjögren's syndrome antigen gene was downregulated. Inflammatory cytokines were also downregulated and growth factors upregulated. This suggests bone marrow cell treatment may successfully repair damaged salivary glands by reducing inflammation and improving cell regeneration and differentiation.
This document discusses using real-time PCR and end-point PCR for gene expression analysis and microarray data verification. Real-time PCR provides a very quantitative and high sensitivity method for determining gene expression levels across a wide dynamic range but requires specialized equipment. End-point PCR provides a less quantitative but still useful method when used with an internal normalizer to control for systematic variation, and does not require specialized equipment. Both methods are useful for direct expression profiling and verification of microarray results on a gene-by-gene basis using commercially available assay kits.
Real-Time quantitative PCR (qPCR) is a mainstream method that is used in research and diagnostic applications for quantification of gene expression. IDT has developed a robust and affordable qPCR master mix for use with probe-based qPCR in single and multiplex assays. In this presentation, we explore a variety of applications of PrimeTime® Gene Expression Master Mix. We cover the use of PrimeTime master mix with probe based assays from IDT. We also look at the use of PrimeTime master mix in multiplex applications without the loss of sensitivity that is commonly observed. Finally, we demonstrate the unmatched stability of PrimeTime master mix under ambient temperatures, saving your research money and minimizing on shipping delays.
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.
Critical Factors for Successful Real-Time PCR: Multiplex PCRQIAGEN
Multiplex end-point PCR is a powerful tool for genotyping and many other applications. QIAGEN’s multiplex PCR chemistry is optimized for reliable amplification of many different templates with high variability in copy numbers. Thus it enables very quick establishment of a new lab routine and instant success for your multiplex PCR strategy.
There is a set of critical factors which we recommend to be regarded for planning and performing this kind of PCR. These will be discussed in detail in the webinar. Additionally, our multiplex PCR chemistry has recently been gaining increasing popularity among scientists who are utilizing it for their next-generation sequencing workflows.
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.
Take your RNA research to the next level with QIAGEN LNA tools!QIAGEN
Download the flyer!
Experience truly exceptional RNA research with QIAGEN's next-generation, LNA®-enhanced tools. LNA (Locked Nucleic Acid) oligos bind with much higher affinity and specificity to RNA targets than standard DNA and RNA oligos – This enables specific and sensitive detection of small RNAs and discrimination between highly similar
sequences.
This document summarizes a presentation about RNase H2 PCR (rhPCR), a new molecular technology that uses an RNA residue in PCR primers and a thermostable RNase H2 enzyme. It describes how rhPCR works, the advantages it provides over traditional PCR including reduced primer-dimer formation and improved specificity for rare allele detection. Two generations of cleavable primer designs - GEN1 and GEN2 - are discussed, along with their different applications. Examples are provided that demonstrate how rhPCR can improve assays for SNP genotyping, multiplex PCR, and detection in complex backgrounds.
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.
Use of CRISPR-Cas9 has revolutionized targeted genome editing. However, rapid design of high-quality guide RNA (gRNA) sequences with high on-target and low off-target editing remains challenging. We implemented a machine learning algorithm to design high-quality gRNA sequences in 5 commonly used species (human, mouse, rat, zebrafish, and nematode). Our tool also designs gRNA sequences against custom targets, and can check existing gRNA designs for quality. In this webinar, we review our data illustrating this tool's performance and demonstrate its use in predicting and designing improved gRNAs for genome editing.
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.
This document provides information about qBiomarker somatic mutation PCR arrays for cancer mutation analysis. It discusses how the arrays can detect mutations in key cancer genes and pathways. The document outlines the workflow, which involves using quantitative PCR to detect mutations in DNA samples. Data is analyzed by comparing Ct values between mutation assays and controls to identify which samples contain mutations. The document provides examples of this analysis method using lung and colon cancer cell line and patient samples.
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 importance of controls and novel solutions for successful real-time qPCRQIAGEN
The increasing demand for streamlined, monitored and ultrafast qPCR procedures requires high-performance, real-time quantitative RT and PCR chemistries. Particularly, procedures utilizing generic kits for gene expression analysis should include in-process safety measures to avoid variables and control accuracy of procedures and results. This slidedeck presents innovative solutions for one-step and two-step RT-PCR that significantly enhance performance and reliability in qRT-PCR. The new QuantiNova kit family offers a combination of various integrated safety features to remove variables and prevent artifacts. Internal control RNA, removal of genomic DNA, room temperature set-up capability for RT-PCR and a built-in visual pipetting control verify accurate procedures, ensuring reliable gene expression profiling.
This slidedeck explains the principles of the technologies and shows data demonstrating performance in qRT-PCR. Find out how you can verify accurate performance in qRT-PCR and improve your results!
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.
Cancer therapies that target specific pathways can be more effective than established, nonspecific chemotherapy and radiation treatments, and may prevent side effects on healthy tissues. Such targeted therapies can only be applied after underlying gene mutations have been identified. However, detecting low frequency variants from clinically relevant samples poses significant challenges. Specimens are routinely formalin-fixed and paraffin-embedded (FFPE) for histology, which can decrease the efficiency of NGS library preparation. In this presentation, we discuss approaches for extraction of DNA from FFPE samples, and recommend quality control assays to guide parameter selection for library construction and sequencing depth.
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.
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
Alzheimer’s disease (AD) is a devastating neurodegenerative disease that is genetically complex. Although great progress has been made in identifying fully penetrant mutations in genes that cause early-onset AD, these still represent a very small percentage of AD cases. Large-scale, genome-wide association studies (GWAS) have identified at least 20 additional genetic risk loci for the more common form: late-onset AD. However, the identified SNPs are typically not the actual risk variants, but are in linkage disequilibrium with the presumed causative variants [1].
To help identify causative genetic variants, we have combined highly accurate, long-read sequencing with hybrid-capture technology. In this collaborative webinar*, we present this method and show how combining IDT xGen® Lockdown® Probes with PacBio SMRT® Sequencing allows targeting and sequencing of candidate genes from genomic DNA and corresponding transcripts from cDNA. Using a panel of target capture probes for 35 AD candidate genes, we demonstrate the power of this approach by looking at data for two individuals with AD. Some additional benefits of this method include the ability to leverage long reads, phase heterozygous variants, and link corresponding transcript isoforms to their respective alleles.
Reference: 1. Van Cauwenberghe C, Van Broeckhoven C, Sleegers K. (2016) The genetic landscape of Alzheimer disease: clinical implications and perspectives. Genet Med, 18(5):421–430.
* This presentation represents a collaboration between Pacific Biosciences and Integrated DNA Technologies. The individual opinions expressed may not reflect shared opinions of Pacific Biosciences and Integrated DNA Technologies.
The CRISPR-Cas9 system has emerged as one of the leading tools for modifying genomes of organisms ranging from E. coli to humans. One of the key components of this editing system is Cas9 endonuclease. The cleavage activity of the S. pyogenes Cas9 enzyme is mediated by the coordinated functions of two catalytic domains and creates blunt-ended, double-stranded breaks. Alanine substitution at key residues within these domains creates two Cas9 nickase variants. Variant D10A produces a nick on the targeting strand, while H840A nicks the non-targeting strand. This double nicking strategy can be leveraged to reduce unwanted off-target effect. However, the nickase experiments can be inherently more complicated than standard CRISPR-Cas9 editing, given the requirement for two guide RNAs to function simultaneously.
In this webinar, both Shuqi Yan and Mollie Schubert present the data from the characterization of a number of factors that impact the efficiency of cooperative nicking in cell cultures. They also summarize a few key design considerations for achieving efficient gene disruption or homology directed repair (HDR) when planning your nickase experiments.
Learn more: http://www.idtdna.com/pages/products/crispr-genome-editing
QIAGEN's qPCR data analysis web portal allows users to analyze quantitative PCR (qPCR) data online. Users can upload raw CT values from SYBR Green or probe-based qPCR assays. The portal will normalize the data, calculate fold changes, and generate graphs and exportable results within 15 minutes. The portal guides users through a seven step process to choose their experiment, upload an Excel file, set up the analysis, view processed results tables and graphs, create additional visualization plots, and download the analyzed data for further exploration. Key benefits include reliable conversion of CT values into fold changes with confidence intervals and p-values, automatic housekeeping gene selection, and suggestions for follow-up experiments.
This study examined the effects of bone marrow cell treatment on salivary glands in mice with Sjögren's syndrome. Researchers extracted mRNA from salivary gland cells of mice with Sjögren's syndrome and used real-time PCR to analyze gene expression. They found that bone marrow cell treatment upregulated genes related to saliva production and downregulated genes associated with inflammation. Specifically, amylase and parotid secretary protein genes were upregulated while the Sjögren's syndrome antigen gene was downregulated. Inflammatory cytokines were also downregulated and growth factors upregulated. This suggests bone marrow cell treatment may successfully repair damaged salivary glands by reducing inflammation and improving cell regeneration and differentiation.
Accelerate Your Discovery with QIAGEN Service Solutions for Biomarker Researc...QIAGEN
This slidedeck will highlight QIAGEN’s service capabilities in sample isolation, microarray and NGS-sequencing, qPCR panel and custom assay development and bioinformatics as we look at the identification of potential biomarkers and gene signatures. The applications of QIAGEN Service Core in microRNA discovery for toxicology markers in serum and plasma and in identification of RNA signatures for tumor stratification are featured. Learn how you can accelerate your research with QIAGEN service solutions.
This document summarizes a presentation given by Dr. Jo Vandesompele on state-of-the-art normalization of RT-qPCR data. It discusses the importance of normalization to remove experimental variation and introduces the geNorm algorithm for determining the optimal number and combination of reference genes for normalization. GeNorm has become the standard method for reference gene validation and normalization and has improved qPCR data analysis. The document also proposes a novel global mean normalization strategy for large-scale gene expression studies.
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.
This document summarizes real-time PCR (qPCR) and its applications. It discusses:
1) The key components and steps of traditional PCR versus real-time PCR, which allows detection of amplified DNA during the reaction rather than at the end.
2) The two main types of real-time PCR - hydrolysis probe-based (e.g. TaqMan) and DNA-binding dye-based (e.g. SYBR Green) - and how they work.
3) Common applications of real-time PCR like gene expression analysis and advantages like increased specificity of hydrolysis probes over DNA-binding dyes.
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.
Introduction to real-Time Quantitative PCR (qPCR) - Download the slidesQIAGEN
This slidedeck introduces the concepts of real-time PCR and how to conduct a real-time PCR assay. The topics that are covered include an overview of real-time PCR chemistries, protocols, quantification methods, real-time PCR applications and factors for success.
The document discusses BioChain's products for PCR and sample preparation, including PCR enzymes, reverse transcriptases, master mixes, dNTPs, and supporting reagents. It provides details on BioChain's Taq DNA polymerase and Hot Start Taq DNA polymerase, which are produced under strict quality control. It also describes BioChain's UltraScript reverse transcriptase, which is ideal for cDNA synthesis of templates with secondary structure or high GC content. Furthermore, it mentions BioChain's pre-mixed master mixes for standard and quantitative PCR, which offer convenience and reproducibility.
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.
This document provides an introduction to real-time quantitative PCR (qPCR). It discusses what qPCR is, how it works, its applications and workflow. Specifically, it explains that qPCR allows for monitoring of PCR reactions during early and exponential phases to quantify initial amounts of target templates. It also outlines common applications like gene expression analysis, discusses important considerations for assay design and optimization, and provides an overview of the basic qPCR workflow from sample preparation to data analysis.
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.
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.
A Field-Deployable Insulated Isothermal PCR-Based System for Rapid and Sensit...Simon Chung - genereach
POCKIT Central PCR System for Detecting African Swine Fever Virus in Vietnam
The document describes a study evaluating the POCKIT Central PCR system for detecting African Swine Fever Virus (ASFV) in Vietnam. The system provides fully automated sample-to-answer detection of ASFV in 85 minutes using cartridges that integrate nucleic acid extraction and PCR. The study found the POCKIT Central system performed equivalently to the OIE reference real-time PCR method, with high analytical sensitivity and specificity. The automated system reduces hands-on time and human error compared to traditional PCR methods. It provides a simple workflow for rapid on-site detection of ASFV to help control the ongoing spread of the disease
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.
1. The document describes a miRNA PCR array experiment to analyze miRNA expression during osteogenesis and neurogenesis differentiation. Samples were collected from human mesenchymal stem cells (hMSCs) at different time points during differentiation and analyzed in triplicate using PCR arrays.
2. The data analysis plan involves first calculating the ΔCt for each gene of interest by normalizing to housekeeping genes. The ΔCts are then averaged for each gene within each sample group. Fold changes between groups will then be determined using the ΔΔCt method to identify differentially expressed miRNAs during differentiation.
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 RT2 Profiler PCR Arrays, which allow for pathway-focused gene expression profiling using real-time PCR. The PCR Arrays contain primer sets for 84 relevant genes, plus controls. They have been shown to have high sensitivity, specificity, and reproducibility. The complete system includes optimized primer assays, master mixes, and a first strand synthesis kit. Researchers can use pre-designed arrays focused on biological pathways or diseases, or customize arrays as needed.
This document discusses RNA interference and provides information about QIAGEN's SureSilencing shRNA plasmids for gene knockdown experiments. It begins with an introduction to RNAi mechanisms and challenges. It then describes QIAGEN's SureSilencing shRNA plasmid solution, which features guaranteed high knockdown efficiency, multiple designs to control off-target effects, and experimental validation. The document reviews the plasmid features, design algorithm, applications and provides a workflow for gene knockdown experiments using the plasmids. It emphasizes the importance of including appropriate controls and validation steps to ensure successful RNAi experiments.
Real-time PCR is a technique that monitors DNA amplification during the PCR process in real-time using fluorescence detection. It allows for both quantification of DNA present and detection of DNA amplification as it occurs. Real-time PCR has advantages over traditional PCR such as higher sensitivity, specificity, and ability to provide quantitative results. It uses sequence-specific DNA probes labeled with fluorescent dyes and quenchers to detect amplification of target DNA sequences. Data analysis can provide both absolute and relative quantification of DNA targets. Real-time PCR has many applications including gene expression analysis, disease diagnosis, and food and environmental testing.
Chromatrap® is able to reduce the time spent in the laboratory by minimising the need for manual handling. By reducing the time spent using a pipette and with no incubation required, it means that results are achieved faster.
Achieve Complete Coverage of the SARS-CoV-2 GenomeCamille Cappello
Utilize multiple overlapping amplicons in a single tube, using a rapid, 2-hour workflow to prepare ready-to-sequence libraries. The PCR1+PCR2 workflow generates robust libraries even from low input quantities of DNA that may be subsequently quantified and normalized with conventional methods such as Qubit® or Agilent Bioanalyzer, or optionally using the included Swift Normalase reagents.
Provides coverage of >99% of the SARS-CoV-2 genome from limited viral titers
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.
1073958 wp guide-develop-pcr_primers_1012Elsa von Licy
methods analyze the exponential phase of individual amplification
1. The document outlines guidelines for developing high-quality real-time PCR primers based on lessons from designing assays for over 14,000 genes.
plots. Regardless of the method, efficiencies between 90-110
2. Key factors in primer design include thermodynamic properties, specificity testing to ensure a single amplicon, and verification of high amplification efficiency and reproducibility.
percent are generally acceptable for accurate analysis by the
3. Wet-bench testing of primers is crucial to validate specificity with single peak melt curves and correct sized products on gels, as well as high efficiency.
∆∆CT method.
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
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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.
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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. EpiTect ChIP qPCR System
A Complete System from Chromatin
ImmunoPrecipitation to Data Analysis
Samuel Rulli, Ph.D.
Applications Scientist
Samuel.Rulli@QIAGEN.com
̣
̣
̣
-1-
Sample & Assay Technologies
2. EpiTect ChIP qPCR System
A Complete System from Chromatin
ImmunoPrecipitation to Data Analysis
Questions, Comments, Concerns?
US Applications Support
Questions, Comments, Concerns?
Global Applications Support
SABio@qiagen.com
888-503-3187
̣
support@sabiosciences.com
*
-2-
Sample & Assay Technologies
4. Webinar Overview
Introduction:
Basic model of Gene Expression, Epigenetics
What is ChiP?
Why ChiP-qPCR?
Research Applications for ChIP PCR Arrays
Summary
-4-
Sample & Assay Technologies
5. Basic Gene Expression Model
Protein “A”
Transcription
Initiation Complex
mRNA ”A”
Structural Gene “A”
-5-
Sample & Assay Technologies
6. The Current Model
Activated
Transcription Factors
RNAi:
shRNA
siRNA
Protein “A”
NFκB
+
p53
Transcription
Initiation Complex
mRNA ”A”
–
Histones
p53 BS Me
Me
Me
Me Me
NFκB BS
DNA Methylation
Ac
Me
Histone-DNA
Interactions
Me Me
Structural Gene “A”
or Reporter system
DNA Methylation
-6-
Sample & Assay Technologies
7. The Current Model
Activated
Transcription Factors
RNAi:
shRNA
miRNA
Protein “A”
NFκB
+
p53
Transcription
Initiation Complex
mRNA ”A”
–
Histones
p53 BS Me
Me
Protein-DNA
Interactions
Me
Me Me
NFκB BS
Ac
Me
Histone-DNA
Interactions
Me Me
Structural Gene “A”
TSS (+1) or Reporter system
DNA Methylation
*
-7-
Sample & Assay Technologies
10. What IS ChIP-qPCR?
What is ChIP?
Chromatin Immuno-Precipitation
Method to examine in vivo protein-DNA interactions
• Transcription Factors, Modified Histones, Other nuclear factors
̣
̣
What is qPCR?
qPCR = Quantitative Real-Time PCR
Faster, Higher Throughput than agarose gel electrophoresis
Gold-Standard Method to Detect DNA
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̣
̣
ChIP-qPCR is Chromatin Immunoprecipitation with qPCR Detection and Quantification
*
- 10 -
Sample & Assay Technologies
11. What IS ChIP-qPCR?
What is ChIP?
Chromatin Immuno-Precipitation
Method to examine in vivo protein-DNA interactions
• Transcription Factors, Modified Histones, Other nuclear factors
̣
̣
What is qPCR?
qPCR = Quantitative Real-Time PCR
Faster, Higher Throughput than agarose gel electrophoresis
Gold-Standard Method to Detect DNA
̣
̣
̣
ChIP-qPCR is Chromatin Immunoprecipitation with qPCR Detection and Quantification
*
- 11 -
Sample & Assay Technologies
15. EpiTect qPCR Primers versus end-point PCR
Cycles
22
26
40
32
Semi-Quantitative PCR
IgG
Ab
Input
IgG
Ab
Input
IgG
Ab
Input
IgG
A
Ab
Input
ChIP-qPCR Primers:
• EVERY Site on EVERY Human,
Mouse, or Rat Promoter
• Specially Designed for Genomic
DNA Enriched from ChIP
• Validated Primer Specificity &
High Amplification Efficiency
B
Ct(dRn)
• Similar Amplification Efficiencies
for Each Primer Pair
Input
Ab
IgG
Assay Design:
• Based on 30 kb promoter tiling
• -20 kb to +10 kb relative to the
TSS
Cycles
Quantitative Real-Time PCR
• Each primer set targets 1 kb
region
- 15 -
Sample & Assay Technologies
17. Applications for ChIP
Where does my Transcription Factor (or any protein that binds DNA) interact with
gDNA?
Example: Which p53 binding site is being used?
What is the chromatin structure (histone modifications)?
Example: Can p53 bind to this site?
Which question you are asking determines the experimental setup:
How many samples? (2 samples or 100?)
Which Antibody?
(is it validated for ChiP?)
Which method for detection? (microarray, sequencing, PCR, qPCR?)
Data Analysis?
Both questions may be necessary to fully understand gene regulation.
- 17 -
Sample & Assay Technologies
18. Challenges Facing Biological Researchers
ChIP Procedure Related
̣
• Time-consuming (multiple days)
• Requires optimization of multiple steps
• Low DNA yields
Antibody Related
̣
• Screening for ChIP-grade suitability tedious & frustrating
DNA Quantification & Analysis Related
̣
•
•
•
•
Low throughput
Requires genomic DNA real-time PCR primer design experience
Requires positive & negative control assays
Tedious data analysis
Reproducible Results are a function of the Biological System and Technique
- 18 -
Sample & Assay Technologies
19. EpiTect System – A Complete Solution
Removing Technique so Researchers can focus on the Biology
?
EpiTect One-Day Kit
̣
One-Day Protocol, Higher DNA Yield and Quality
Cross-Link
EpiTect-Grade Antibody Kits
̣
Validated ChIP-Grade Antibodies + Positive & Negative Controls
Sonication
EpiTect qPCR Arrays
̣
Multiple Assays & Controls Arranged in ONE PCR Plate
96 or 384 well Format Compatible with Most qPCR Instruments
Gene Regulation by Histone Modification
Custom Options Available
ChIP
EpiTect-qPCR Primers
̣
Primers for Every Site on Every Human, Mouse, or Rat Promoter
Validated & Wet-Bench Tested
Optimized SYBR Green Master Mix
̣
̣
Data Analysis Software (Excel template)
qPCR
Data
Analysis
Answer
- 19 -
Sample & Assay Technologies
20. Research Application Example I:
What is the mechanism of CDKN1A expression after 5-FU treatment?
5-FU treatment increases the expression of CDKN1A
Activates p53 transcription Factor
CDKN1A has p53 biniding sites (4 consensus sites!)
Research Questions:
Does p53 bind to the CDKN1A promoter?
Which of the 4 sites are used?
Are all 4 sites equally accessible? (chromatin strucutre questions)
How can we experimentally accomplish this?
- 20 -
Sample & Assay Technologies
24. EpiTect Antibody Kits
For TF and Positive Controls
ChIP-Grade TF Antibody Kits
RNA Pol II
p53
Benefits:
• Experimentally Validated, Ready-to-Use
ChIP-Grade Antibodies & Control IgG
• High Efficiency and Low Background
• Includes Appropriate Positive & Negative
Control Real-Time qPCR Primers
The EpiTect ChIP qPCR System is compatible with any “ChIP grade” antibody
- 24 -
Sample & Assay Technologies
26. EpiTect qPCR Assay Design: Tiling Scheme
TSS (+1)
Structural Gene
mRNA
gDNA
Distal Promoter
1 kb
Proximal Promoter
̣
1 kb
1 kb
qPCR Assay & Amplicon
1 kb
1 kb
Amplicon Coverage Range (Tile)
Thirty (30) 1-kb tiles from -20 kb to +10 kb relative to each TSS
Accommodate every human, mouse and rat gene promoter
Balance Between Sensitivity & Resolution
Quality Controlled for Specificity & Amplification Efficiency
̣
̣
- 26 -
Sample & Assay Technologies
27. Wet Bench QC Validation of ALL PCR Arrays
All PCR Arrays are Wet-Bench Validated
• Assays in each PCR Array are tested for
•Sensitivity:
-constant C(t) value for same amount of template
•Amplification Efficiency:
-single curve analysis and standard curve dilutions
•Specificity:
-Single: single melt-peak during dissociation
- 27 -
Sample & Assay Technologies
28. Finding Your ChIP-qPCR Assays
EVERY Binding Site on EVERY Human, Mouse, Rat Promoter
Search by Gene
• Find primers for known or predicted nuclear factor binding sites relative to TSS
̣
Search by Transcription Factor and/or Gene
• Find primers for binding sites on transcription factor-targeted genes or predicted
binding sites in genes of interest
̣
Search by Chromosomal Position
• Find primers for specific locations from ChIP-on-chip or ChIP-Seq
̣
http://www.sabiosciences.com/chipqpcrsearch.php
- 28 -
Sample & Assay Technologies
29. How the ChIP PCR Array System Works: Summary
• ChIP Fraction Preparation
• One-Day Kit & Antibody Kits
• 6 hours
• Load Plates (Preferably with
multi-channel or repeating
pipettors)
• 2 minutes
• Run 40 cycle qPCR Program
• 2 hours
• Upload and Analyze Data
• 15 minutes
- 29 -
Sample & Assay Technologies
31. Experimental Treatments Alter Transcription
Factor Binding
Treatment with 5-FU increases p53 Binding to CDKN1A Promoter by 5- to 9-fold.
It also increases RNA Pol II binding to CDKN1A Promoter by 7.5-fold.
- 31 -
Sample & Assay Technologies
32. Data Analysis
Data Analysis Principles:
Normalize ChIP Fraction Ct to Input DNA Fraction Ct Values
Percent of Input (or fraction enrichment) = 100 * 2 ^ -∆Ct
∆
Fold-Changes in DNA Binding = 2 ^ -∆∆ t
∆∆C
∆∆
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̣
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Free Data Analysis Excel Template:
http://www.sabiosciences.com/manuals/chipqpcranalysis.xls
- 32 -
Sample & Assay Technologies
33. Are all of the p53 sites equally accessible?
P53 can interact with the -12, -3, -2 tiles but not -4
Is the chromatin structure different?
- 33 -
Sample & Assay Technologies
35. EpiTect Antibody Kits
For Histone Modifications
ChIP-Grade Histone Antibody Kits
Antibody Name
Modifications
H3ac
H3K9ac
Acetylation
Benefits:
H4ac
H3K4me1
• Experimentally Validated, Ready-to-Use
ChIP-Grade Antibodies & Control IgG
H3K4me2
H3K4me3
• High Efficiency and Low Background
H3K9me1
H3K9me2
H3K9me3
Methylation
• Includes Appropriate Positive & Negative
Control Real-Time qPCR Primers
H3K27me3
H3K36me3
H3K79me3
H4K20me3
H3
Unmodified Control
- 35 -
Sample & Assay Technologies
36. How the ChIP PCR Array System Works: Summary
• ChIP Fraction Preparation
• One-Day Kit & Antibody Kits
• 6 hours
• Load Plates (Preferably with
multi-channel or repeating
pipettors)
• 2 minutes
• Run 40 cycle qPCR Program
• 2 hours
• Upload and Analyze Data
• 15 minutes
- 36 -
Sample & Assay Technologies
37. Modified Histone Distribution Across a Promoter
-1
Histone markers for actively transcribed genes (H3Ac and H3K4me2) but not for
transcriptionally inactive genes (H3K27me3) surround the CDKN1A gene making it
available to p53 regulation.
- 37 -
Sample & Assay Technologies
38. Application Example 2: Chromatin structure for a pathway
Are changes in gene expression caused by Epigenetic Changes?
DNA Methylation
Chromatin Structure
miRNA
Methyl-Profiler
PCR Array
Epigenetic ChIP
PCR Array
miRNA
PCR Array
- 38 -
Sample & Assay Technologies
40. How the ChIP PCR Array System Works: Summary
• ChIP Fraction Preparation
• One-Day Kit & Antibody Kits
• 6 hours
• Load Plates (Preferably with
multi-channel or repeating
pipettors)
• 2 minutes
• Run 40 cycle qPCR Program
• 2 hours
• Upload and Analyze Data
• 15 minutes
- 40 -
Sample & Assay Technologies
41. EpiTect PCR Arrays
for Histone Regulation of Gene Expression
Analyze Pathway-Focused in vivo Protein-DNA Interactions
Using ChIP-enriched genomic DNA, simultaneously analyze DNAprotein interactions across a focused panel of genes representing an
epigenetically controlled biological pathway or disease state
̣
Evaluate association of histones or modified histones interacting with a
genomic region known for active transcriptional regulation
̣
Each array is a 96 or 384-well PCR plate containing experimentally
validated PCR primers (84 pathway-focused genes, 12 controls) that
target the proximal promoter region (+1 kb from the TSS) of each
gene
̣
̣
Custom EpiTect qPCR Arrays Available
- 41 -
Sample & Assay Technologies
43. Modified Histone Distribution Across a Promoter
Indication of OPEN chromatin and Transcription
-1
Histone markers for actively transcribed genes (H3Ac and H3K4me2) but not for
transcriptionally inactive genes (H3K27me3) surround the TSS
- 43 -
Sample & Assay Technologies
44. EpiTect ChIP qPCR Array Layout
1
2
3
4
5
6
7
8
A
CDX2
DACH1
DLX1
DLX2
DNMT3B
EGR#
ESR1
EZH2
B
FOXP3
GATA1
GATA6
GLI2
C
HOXB13
HOXB3
HOXB5
D
HOXD4
HTR7
IRX4
E
NEUROD1
NFATC1
NKX2-2 NOTCH2
F
PITX3
POU4F1
POUF2
G
SP1
STAT1
STAT3
H
10
12
FOXA1 FOXA2 FOXP1 FOXP2
HOXC4
HOXC5 HOXC6 HOXC9 HOXD1 HOXD10
JUN
KLF2
KLF4
NRF2
OLIG2
PAX1
PAX5
POU5F1
PPARG
RB1
RUX1
SIX2
TBX5
TDGF1
TERT
TLX3
VDR
WRN
MYOD1 SERPINA1 SAT2
SAT
GAPDH(-3) GAPDH (+1) GAPDH+4 ALDOA
11
HAND1 HOXA10 HOXA11 HOXA2 HOXA3 HOXA7 HOXA9 HOXB1
HOXB8 HOXC10 HOXC12
ISL1
9
RPL30
Transcriptionally Active Genes
Transcriptionally Inactive Genes
Silenced Heterochromatin
Positive PCR Control
LIN28B LMX1B MSX2
MYC
NANOG
PAX9
PCNA
PITX2
SMAD2 SOX2
SOX6
SOX9
WT1
ZFPM2
ZIC1
IGX1A
PPC
PPC
PAX6
GAH-501
Human Stem Cell
Transcription Factors
Control primer sets include:
• System control to assess the qPCR performance (machine & handling).
• Different chromatin regions representing active and inactive chromatin regions to
evaluate the reliability of genomic DNA samples enriched by ChIP.
- 44 -
Sample & Assay Technologies
45. EpiTect ChIP qPCR Array Layout
1
3
4
5
6
7
8
A
CDX2
DACH1
DLX1
DLX2
DNMT3B
EGR#
ESR1
EZH2
B
FOXP3
GATA1
GATA6
GLI2
C
HOXB13
HOXB3
HOXB5
D
HOXD4
HTR7
IRX4
E
NEUROD1
NFATC1
NKX2-2 NOTCH2
F
PITX3
POU4F1
POUF2
G
+1
Tile
ONLY
2
SP1
STAT1
STAT3
H
10
12
FOXA1 FOXA2 FOXP1 FOXP2
HOXC4
HOXC5 HOXC6 HOXC9 HOXD1 HOXD10
JUN
KLF2
KLF4
NRF2
OLIG2
PAX1
PAX5
POU5F1
PPARG
RB1
RUX1
SIX2
TBX5
TDGF1
TERT
TLX3
VDR
WRN
MYOD1 SERPINA1 SAT2
SAT
GAPDH(-3) GAPDH (+1) GAPDH+4 ALDOA
11
HAND1 HOXA10 HOXA11 HOXA2 HOXA3 HOXA7 HOXA9 HOXB1
HOXB8 HOXC10 HOXC12
ISL1
9
RPL30
Transcriptionally Active Genes
Transcriptionally Inactive Genes
Silenced Heterochromatin
Positive PCR Control
LIN28B LMX1B MSX2
MYC
NANOG
PAX9
PCNA
PITX2
SMAD2 SOX2
SOX6
SOX9
WT1
ZFPM2
ZIC1
IGX1A
PPC
PPC
PAX6
GAH-501
Human Stem Cell
Transcription Factors
Control primer sets include:
• System control to assess the qPCR performance (machine & handling).
• Different chromatin regions representing active and inactive chromatin regions to
evaluate the reliability of genomic DNA samples enriched by ChIP.
- 45 -
Sample & Assay Technologies
46. EpiTect ChIP qPCR Arrays for Histone Regulation
of Gene Expression
Human and Mouse Cataloged Arrays Available:
Stem Cell Transcription Factors
Oncogene & Tumor Suppressor Genes
T Helper Cell Differentiation
Inflammatory Responses
Cancer Drug Targets
DNA Repair
Cell Cycle
Cell Lineage Identification
Polycomb & Trithorax Genes
Polycomb & Trithorax Complexes
Homeobox (HOX) Genes
̣
Custom EpiTect PCR Arrays
Promoter Binding Arrays – examine the promoter region of a particular gene
Gene Regulation Arrays – Follow up to ChIP-on-chip or ChIP-Seq
̣
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Sample & Assay Technologies
47. EpiTect ChIP qPCR Arrays for Histone Regulation
of Gene Expression
Human and Mouse Cataloged Arrays Available:
Stem Cell Transcription Factors
Oncogene & Tumor Suppressor Genes
T Helper Cell Differentiation
Inflammatory Responses
Cancer Drug Targets
DNA Repair
Cell Cycle
Cell Lineage Identification
Polycomb & Trithorax Genes
Polycomb & Trithorax Complexes
Homeobox (HOX) Genes
̣
Custom EpiTect PCR Arrays
Promoter Binding Arrays – examine the promoter region of a particular gene
Gene Regulation Arrays – Follow up to ChIP-on-chip or ChIP-Seq
̣
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Sample & Assay Technologies
49. Stem Cell Research Application Detail
____
____ ____
____
____
0 48048048 048048
_________ _________
Day
0
4
8 0
4
________
8 0 4
________
8 0 4
8
________
0
4 8
Nanog
Oct4
Sox2
Dlx1
Zfpm2
Neurod1
Hoxa3
Heat map visualization of the dynamic change
in pluripotency-associated gene expression
and histone modification during differentiation.
•H3Ac and H3K4me3 – transcriptionally active euchromatin
•H3K27me – transcriptionally inactive euchromatin
•H3K29me3 – silenced gene, densely packed heterochromatin
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Sample & Assay Technologies
50. Summary of the EpiTect System
?
EpiTect One-Day Kit
̣
One-Day Protocol, Higher DNA Yield and Quality
Cross-Link
EpiTect-Grade Antibody Kits
̣
Validated ChIP-Grade Antibodies + Positive & Negative Controls
Sonication
EpiTect PCR Arrays
̣
Multiple Assays & Controls Arranged in ONE PCR Plate
96 or 384 well Format Compatible with Most qPCR Instruments
Gene Regulation by Histone Modification
Custom Options Available
ChIP
EpiTect-qPCR Primers
̣
Primers for Every Site on Every Human, Mouse, or Rat Promoter
Validated & Wet-Bench Tested
Optimized SYBR Green Master Mix
̣
̣
Data Analysis Software (Excel template)
qPCR
Data
Analysis
Answer
- 50 -
Sample & Assay Technologies
52. EpiTect ChIP qPCR Arrays for Histone Regulation
of Gene Expression
Human and Mouse Cataloged Arrays Available: (+1 TSS qPCR Assay for many Genes)
Apoptosis
Cell Cycle
DNA Repair
Homeobox (HOX) Genes
Polycomb & Trithorax Genes
Polycomb & Trithorax Complexes
T Helper Cell Differentiation
Cancer Drug Targets
Cell Lineage Identification
EMT (Epithelial to Mesenchymal Transition)
Inflammatory Responses
Oncogene & Tumor Suppressor Genes
Stem Cell Transcription Factors
Toll-like Receptor Signaling
Custom EpiTect ChIP qPCR Arrays
Promoter Binding Arrays – examine the promoter region of a particular gene
Gene Regulation Arrays – Follow up to ChIP-on-chip or ChIP-Seq
̣
̣
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Sample & Assay Technologies
53. EpiTect One-Day Kit Benefits
Speed & Ease
̣
Cross-Link
• One-day protocol
• Minimal steps to optimize
Universality
Sonication
̣
• Compatible with ANY ChIP-Grade antibody
Reliability & Consistency
̣
• Highly reproducible in different researchers’ hands
• qPCR quality DNA
ChIP
qPCR
Data
Analysis
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Sample & Assay Technologies
54. Solutions Provided by EpiTect qPCR System
Pathway, Disease or single loci Analysis using qPCR
• Genome wide qPCR Assays around every TSS in Human, Mouse
Rat genome
• Custom PCR Arrays can be manufactured to simultaneously
analyze up to 96 or 384 genomic loci
• Complement & follow-up for ChIP-on-chip & ChIP-Seq
̣
Reliable & Sensitive
• Stringent real-time PCR primer QC procedure
• As little as one million cells per ChIP assay (25-50 µg chromatin)
̣
Easy to Use:
• Simplified ChIP preparation protocol
• Accessible to any lab with access to qPCR instrument
• Simplified Data Analysis templates
̣
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Sample & Assay Technologies
55. ChampionChIP qPCR System™
A Complete System from Chromatin Precipitation to Data Analysis
Outside US? Email SABio@qiagen.com for discount offers in your region.
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Sample & Assay Technologies
56. SciGlobe: Search a for your papers using a bigger
database than Pubmed and more relevant than Google Scholar
SciGlobe Advantage
•Free
•Weekly emails on
new papers
•Download and store
papers
•Export to citation
databases
•Larger database than
pubmed
•More relevant
searches than Google
scholar
Try it Now: http://www.sabiosciences.com/sciglobe.php
- 56 -
Sample & Assay Technologies
57. EpiTect qPCR System™
A Complete System from Chromatin Precipitation to Data Analysis
Questions, Comments, Concerns?
US Applications Support
Questions, Comments, Concerns?
Global Applications Support
Discount offers? Register:
SABio@qiagen.com
888-503-3187
̣
support@sabiosciences.com
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Sample & Assay Technologies