This document discusses tools for studying inflammation regulation and gene expression. It describes how QIAGEN offers products for various experimental techniques in inflammation research, including gene expression analysis using RT-PCR and real-time PCR. Case studies are presented on studying the roles of transcription factors and miRNAs in T-cell maturation and inflammation resolution, as well as analyzing DNA methylation changes related to aberrant gene expression in a vasculitis disease. The document provides an overview of experimental designs and results using QIAGEN products such as PCR arrays to examine differential gene and miRNA expression during inflammation.
Watch the presentation of this webinar here: https://bit.ly/2SWCycq
mRNA has taken center stage. Vaccines and therapeutics based on this versatile biomolecule have the potential to transform disease prevention and treatment. This webinar will explore key considerations for efficient mRNA production, starting from facility design and raw materials selection to technologies and strategies used for manufacturing.
The success of mRNA-based COVID-19 vaccines has created a significant level of interest in this versatile biomolecule for disease prevention and treatment. While production of these vaccines took place in record time, critical decisions must be made when developing novel mRNA applications to ensure manufacturability, reproducibility, and safety. This webinar will explore foundational elements of the mRNA manufacturing workflow and strategies to design the right facilities to ensure success. Topics include collaborative approaches to ensure access to high quality raw materials, application of advanced technologies for manufacturing, options for facility design and key considerations when leveraging a contract development and manufacturing partner.
In this webinar, you will learn:
• Therapeutic potential of mRNA: COVID-19 and beyond
• How mRNA manufacturing workflows and facility design have a significant impact on reproducibility and performance
• Amptec capabilities to accelerate mRNA development and manufacturing
Single-cell microRNA expression profiling is a challenging workflow. From cell lysis, reverse transcription, preamplificatin to real-time PCR, every step involves technical pitfalls. Therefore it is critical to have a robust system that facilitates universal cDNA synthesis and universal amplification of all miRNAs in one workflow without introducing bias. Here we present a new poster – introducing a robust real-time PCR workflow and protocol for profiling miRNA expression from a single cell and how we analyze the single cells by using the free data analysis software.
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.
Data driven strategies and considerations for scalable purification of Plasmi...Merck Life Sciences
The document discusses strategies and considerations for the scalable purification of plasmid DNA for use in vaccine manufacturing. It covers the key upstream steps of cell harvest, lysis, and clarification. Cell harvest is typically done using centrifugation or tangential flow filtration. Lysis is usually an alkaline lysis process that must be carefully optimized to avoid damaging the plasmid DNA. Clarification can involve various pretreatment and filtration steps to reduce impurities before downstream purification. No single platform solution currently exists and processes vary between manufacturers.
This document describes a study that evaluated the use of multiple displacement amplification (MDA) for whole-genome amplification in preimplantation genetic diagnosis (PGD) of Marfan syndrome. The researchers found that MDA allowed analysis of five genetic loci from single cells. Using MDA, they developed a PGD protocol for Marfan syndrome and diagnosed seven embryos, transferring two healthy embryos that resulted in an ongoing pregnancy and healthy birth. The study demonstrates that MDA is useful for overcoming the challenge of limited DNA in PGD and allows diagnosis of any known genetic defect using standard methods.
On March 14 I presented the history of my research activities and proposals for MS Biology thesis work for the students entering the program at National University,
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
Purification of total RNA from peripheral blood mononuclear cells - Download ...QIAGEN
Peripheral blood is often used for in vitro studies of the human immune system or immune responses, such as inflammation. An important part of the human immune system is represented by the peripheral blood mononuclear cells (PBMC). PBMC are blood cells characterized by a round nucleus and consist mainly of lymphocytes (T cells, B cells, and NK cells), macrophages and dendritic cells. Here, we describe the analysis of lipopolysaccharide-induced transcriptional response of isolated PBMC from whole blood using the RNeasy® Mini Kit or RNeasy Micro Kit, RT2 First Strand Kit, RT2 SYBR® Green ROX™ qPCR Mastermix, and RT2 Profiler PCR Arrays.
Watch the presentation of this webinar here: https://bit.ly/2SWCycq
mRNA has taken center stage. Vaccines and therapeutics based on this versatile biomolecule have the potential to transform disease prevention and treatment. This webinar will explore key considerations for efficient mRNA production, starting from facility design and raw materials selection to technologies and strategies used for manufacturing.
The success of mRNA-based COVID-19 vaccines has created a significant level of interest in this versatile biomolecule for disease prevention and treatment. While production of these vaccines took place in record time, critical decisions must be made when developing novel mRNA applications to ensure manufacturability, reproducibility, and safety. This webinar will explore foundational elements of the mRNA manufacturing workflow and strategies to design the right facilities to ensure success. Topics include collaborative approaches to ensure access to high quality raw materials, application of advanced technologies for manufacturing, options for facility design and key considerations when leveraging a contract development and manufacturing partner.
In this webinar, you will learn:
• Therapeutic potential of mRNA: COVID-19 and beyond
• How mRNA manufacturing workflows and facility design have a significant impact on reproducibility and performance
• Amptec capabilities to accelerate mRNA development and manufacturing
Single-cell microRNA expression profiling is a challenging workflow. From cell lysis, reverse transcription, preamplificatin to real-time PCR, every step involves technical pitfalls. Therefore it is critical to have a robust system that facilitates universal cDNA synthesis and universal amplification of all miRNAs in one workflow without introducing bias. Here we present a new poster – introducing a robust real-time PCR workflow and protocol for profiling miRNA expression from a single cell and how we analyze the single cells by using the free data analysis software.
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.
Data driven strategies and considerations for scalable purification of Plasmi...Merck Life Sciences
The document discusses strategies and considerations for the scalable purification of plasmid DNA for use in vaccine manufacturing. It covers the key upstream steps of cell harvest, lysis, and clarification. Cell harvest is typically done using centrifugation or tangential flow filtration. Lysis is usually an alkaline lysis process that must be carefully optimized to avoid damaging the plasmid DNA. Clarification can involve various pretreatment and filtration steps to reduce impurities before downstream purification. No single platform solution currently exists and processes vary between manufacturers.
This document describes a study that evaluated the use of multiple displacement amplification (MDA) for whole-genome amplification in preimplantation genetic diagnosis (PGD) of Marfan syndrome. The researchers found that MDA allowed analysis of five genetic loci from single cells. Using MDA, they developed a PGD protocol for Marfan syndrome and diagnosed seven embryos, transferring two healthy embryos that resulted in an ongoing pregnancy and healthy birth. The study demonstrates that MDA is useful for overcoming the challenge of limited DNA in PGD and allows diagnosis of any known genetic defect using standard methods.
On March 14 I presented the history of my research activities and proposals for MS Biology thesis work for the students entering the program at National University,
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
Purification of total RNA from peripheral blood mononuclear cells - Download ...QIAGEN
Peripheral blood is often used for in vitro studies of the human immune system or immune responses, such as inflammation. An important part of the human immune system is represented by the peripheral blood mononuclear cells (PBMC). PBMC are blood cells characterized by a round nucleus and consist mainly of lymphocytes (T cells, B cells, and NK cells), macrophages and dendritic cells. Here, we describe the analysis of lipopolysaccharide-induced transcriptional response of isolated PBMC from whole blood using the RNeasy® Mini Kit or RNeasy Micro Kit, RT2 First Strand Kit, RT2 SYBR® Green ROX™ qPCR Mastermix, and RT2 Profiler PCR Arrays.
Detection and Surveillance of Antibiotic Resistance Genes From Food and Ferti...QIAGEN
One potential way to acquire antibiotic resistance genes is through the food supply chain. Both livestock and feed may
acquire antibiotic resistant bacteria via different mechanisms. Foodstuffs can be exposed to antibiotic resistant bacteria
through fertilizer originating from waste-water treatment plants. This, in addition to increasing administration of antibiotics
to livestock, can lead to food being a potential source of antibiotic resistance genes. This may lead to horizontal gene
transfer to pathogenic enteropathogens and further to drug resistance in humans. Therefore, the surveillance and prevention
of antibiotic resistance genes in food is important.
To effectively combat the spread of difficult-to-treat bacterial infections, rapid surveillance methods to detect antibiotic
resistance genes are required; in order to monitor both bacterial isolates and metagenomic samples.
Since the gut is known to act as a reservoir for antibiotic resistance genes, a small-scale research study was performed on
5 stool samples isolated from healthy human adults using an antibiotic resistance gene identification PCR array. In addition,
the diversity of antibiotic resistance genes in municipal biosolids was determined using an Antibiotic Resistance Genes
Microbial DNA qPCR Array with DNA extracted from belt-filter, press-cake sewage samples.
22 antibiotic resistance genes were identified from different resistance classifications. Further studies were performed in
beef, chicken, vegetable and pork samples. In conclusion, PCR arrays can be effective tools for detection of antibiotic
resistance genes from food samples and potential fertilizer sources.
This document summarizes tools and techniques for studying the innate immune system. It describes the differences between innate and adaptive immunity, the phases and cellular components of the innate response, pattern recognition receptors, signaling pathways, and technologies for research including PCR arrays and reporter assays. An example is given of using a PCR array to study the role of TLR3 in the response to Chlamydia infection, finding that TLR3 and IFN-β are major mediators. Another example shows using a reporter array to identify NF-κB and other pathways activated by cytokine stimulation.
The document discusses genetically modified (GM) crops and methods for detecting them. It begins with an introduction to GM crops, noting that they are plants modified using genetic engineering to introduce new traits, and that global GM crop acreage has increased significantly in recent decades. It then provides details on three main analytical approaches for detecting GM crops: detection to determine if a product is GM or not, identification of the specific GM crop or trait present, and quantification of GM content. Several DNA-based, protein-based, and trait-based methods are described, including polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), and lateral flow tests. The document compares the different methods and concludes with a discussion of the
Deeper Insight into Transcriptomes! Download the FlyerQIAGEN
Discover a new workflow for RT-PCR-based gene expression work
Accurate and biologically relevant results in RT-PCR-based
gene expression can be difficult to achieve. Successful
transcriptome work requires validated, reproducible targets
and high-quality technology. Recognizing the variability arising from sample physiology
and pathology, the influence of sample purification and
assay conditions, and the importance of access to easyto-
use software, QIAGEN experts developed a new gene
expression workflow. It will help you properly validate your
RT-PCR and gain the deepest insight into your result.
The document summarizes qBiomarker Somatic Mutation PCR Arrays, which are PCR-based assays that can rapidly and accurately detect somatic mutations in cancer samples. The arrays can detect mutations present at as little as 0.01% of DNA in a sample. They have been validated to work reliably with different sample types, including archived samples. The arrays cover a wide range of clinically relevant cancer mutations and allow screening of many mutations simultaneously in a single PCR run. The assays and content were selected based on published data on mutation frequencies and functional significance. The arrays provide a simple method for sensitive somatic mutation profiling to aid cancer research.
Extending miRQC’s dynamic range: amplifying the view of Limiting RNA samples ...QIAGEN
The original microRNA quality control (miRQC) study provided an in-depth analysis of commercially available microRNA (miRNA) quantification platforms. Specifically, twelve different
microarray, real-time PCR and small RNA sequencing platforms were assessed for reproducibility, sensitivity, accuracy, specificity and concordance of differential expression using a variety of sample types. Overall, each platform exhibited specific strengths and weaknesses, leading to the
final suggestion that a platform should be chosen on the basis of the experimental setting and the specific research questions. With this suggestion in mind, and the fact that liquid miRNA biopsies are an area of intense interest, we sought to expand the original miRQC study. For our “miRQC extension,” we benchmarked the QIAGEN miScript® PCR System with and without preamplification, and included a specific focus on routinely used biofluids. Concurrently, we benchmarked the miScript PCR System against another SYBR® Green miRNA detection platform. Overall, QIAGEN miScript demonstrated strong reproducibility and accuracy as well as superior detection rate and sensitivity in biofluids. Collectively, QIAGEN miScript provides the leading solution for novel miRNA discoveries.
Stratified Medicine in Cancer: The Role of HistopathologistDr. Shubhi Saxena
This document discusses stratified medicine approaches for cancer treatment. It describes how cellular pathologists classify gene mutations in cancer, including whether they are germline or somatic, synonymous or non-synonymous, activating or inactivating. Certain mutations can predict treatment response or resistance. Key driver mutations are discussed for lung cancer, including EGFR mutations and ALK translocations. EGFR mutant cancers may respond to EGFR tyrosine kinase inhibitors, while ALK rearrangements are targeted by crizotinib. Histopathology plays an important role in mutation detection and molecular testing to guide targeted therapies.
RNA profiling is a powerful technique for understanding cellular origins and disease states. Recent studies in a variety of diseases have revealed RNA signatures that are excellent biomarker candidates for understanding disease status and predicting progression.
Suppose you want to discover a biomarker. What are the major steps in discovering a biomarker when you start from a blood sample? Here is the story of a researcher who is trying to find blood-based biomarkers in autism spectrum disorders.
Toll-like Receptors in Inflammation: Host Defense Webinar Series Part 2QIAGEN
Toll-like receptors (TLRs) play an important role in the innate immune system and inflammation. TLRs recognize pathogen-associated molecular patterns and activate signaling pathways that induce inflammatory responses. This webinar discusses TLR signaling and the role of TLRs in inflammation, including their association with various diseases. It also explores how long non-coding RNAs and microRNAs help regulate TLR signaling pathways and the inflammatory response. The webinar promotes QIAGEN tools for studying TLRs, such as RT-PCR arrays, that can help profile gene and RNA expression changes related to TLR signaling.
- WPE-stem cells, a type of human prostate progenitor/stem cell, were initially highly sensitive to cadmium exposure, experiencing 90% cell loss after one week compared to 15% for a differentiated cell line. However, the stem cells quickly adapted and recovered their numbers within 4 weeks.
- During chronic cadmium exposure, the stem cells acquired characteristics of cancer cells like increased MMP-9 levels and colony formation along with decreased expression of tumor suppressors. They also showed signs of epithelial-mesenchymal transition.
- The initial sensitivity was linked to increased apoptosis factors while the later adaptation involved upregulation of cadmium resistance factors like metallothionein and downregulation of cadmium
miRNA profiling from blood challenges and recommendations - Download the articleQIAGEN
The discovery of stable miRNA species circulating in blood has led to increased research focus on disease-related variations in serum and plasma miRNA expression and the possibility that such variations could serve as noninvasive biomarkers for disease. Working with serum and plasma miRNA presents various challenges in purification and characterization. In this paper, we outline QIAGEN recommendations for robust purification and quantification, as well as reliable data normalization and analysis.
The document describes the development of two TaqMan digital PCR liquid biopsy assays to detect mutations in the TERT promoter region associated with cancer. The assays target the C228T and C250T mutations. Testing showed the assays could reliably detect the mutations at levels as low as 0.1% mutation frequency in genomic DNA samples. Assay design was challenging due to the high GC content and repetitive sequences in the TERT promoter region. Optimization of thermal cycling conditions was needed for accurate detection of the mutations by digital PCR.
Meeting the challenges of miRNA research: miRNA and its Role in Human Disease...QIAGEN
This document discusses a 4-part webinar series on microRNA (miRNA) research presented by QIAGEN. Part 1 will cover miRNA profiling from biofluids, part 2 will discuss challenges in miRNA research, part 3 will focus on advanced miRNA expression analysis, and part 4 will analyze functional analysis of miRNA. The document provides background on miRNAs and their role in gene expression and disease. It also describes QIAGEN products and solutions for miRNA sample preparation, real-time PCR, data analysis, and functional validation to help researchers overcome challenges in miRNA analysis.
Streamlined next generation sequencing assay development using a highly multi...Thermo Fisher Scientific
Next generation sequencing (NGS) assay development for solid tumor sequencing requires characterization of variant calling directly from formalin-fixed paraffin embedded (FFPE) tissue samples. However, cell line based FFPE and human FFPE samples only contain 2 to 20 variants, which require laboratories to invest significant resources in sample sourcing and preparation when developing assays to detect 100+ variants
PREDICTION OF ANTIMICROBIAL PEPTIDES USING MACHINE LEARNING METHODSBilal Nizami
Increasing resistance toward the conventional antibiotics has become a global concern. Antimicrobial peptides (AMPs) are potential alternatives for conventional antibiotics. Due to cost related reasons in designing and synthesis of AMPs. Machine learning based prediction tools are indispensable.
1) Researchers identified differential methylation of the GRIN2D gene in colon cancer cells using reduced representation bisulfite sequencing, finding it demethylated in approximately 60% of cell lines. 2) They confirmed this epigenetic alteration led to upregulated GRIN2D expression and assessed its role in the cancer phenotype. 3) Knockdown of GRIN2D expression in demethylated RKO colon cancer cells using shRNA corresponded to reduced cell growth, suggesting GRIN2D demethylation contributes to the tumor cell phenotype. Future replication of these findings is needed to fully understand the significance.
Antimicrobial peptides from Croaker FishAjit Antony
The document describes a study on antimicrobial peptides extracted from the croaker fish, Johnius dussumieri. The objective was to extract and characterize antimicrobial peptides from J. dussumieri. Peptides were extracted using acetic acid-acetone precipitation and tested against various gram-positive and gram-negative bacterial strains. Active fractions were purified using solid phase extraction and cation exchange chromatography. The purified fractions showed inhibition against test strains including Staphylococcus aureus, Bacillus cereus, and Vibrio alginolyticus. The study characterized antimicrobial peptides from J. dussumieri that have potential antimicrobial properties.
Maximizing PCR and RT-PCR Success - Download the BrochureQIAGEN
The invention of the polymerase chain reaction (PCR) by K. Mullis and coworkers in 1985 revolutionized molecular biology and molecular medicine. Major research areas, such as biomarker discovery, gene regulation and cancer research are challenging today’s PCR technologies with more demanding requirements. These include the need for increased throughput while reducing costs, higher assay sensitivity and reliable data normalization. Assay development and evaluation, reproducibility of data and time to result are still major problems encountered by researchers.
Meeting today’s challenges in PCR requires advances in all methods of the workflow that starts with sample collection, sample stabilization, and nucleic acid purification, and ends with amplification and detection. The following pages focus on the importance of amplification in meeting these challenges.
Detection and quantification of mutant alleles in tumor tissue allow for research disease monitoring and the research of drug efficacy. Detection of emerging secondary mutations in the same tumor tissue causing resistance to potential treatment will help guide decisions on future treatment plans. Testing for the presence of mutations in cell free DNA (cfDNA) is a less invasive research method than using tumor tissue. We created a research tool for mutation detection at a sensitivity level of 1% and below. This allows researchers to find correlation between types of mutations and types of tumors and determination of potential secondary mutations.
The tool combines TaqMan® SNP Genotyping Assays with digital PCR. A set of assays was optimized for use
in digital PCR with the QuantStudio® 3D Digital PCR System. In digital PCR, partitioning the sample into many individual reaction wells facilitates detection and quantification of rare mutant alleles. TaqMan® SNP Genotyping Assays ensure reliable discrimination of mutant and wild-type allele. Our current set of 60 assays covers mutations commonly found in tumor tissues, such as: BRAF V600E, mutations in EGFR exons 19, 20 and 21, KRAS codons 12 and 13, PIK3CA exons 9 and 20, and the JAK2 V617F mutations. All assays were wet-lab tested at a 10% mutation rate and a 1% mutation rate using mutant plasmid spiked into wild-type genomic DNA. Additionally, selected assays were tested at the 0.1% mutation rate using mutant cell lines spiked into wild-type genomic DNA. Wet-lab results confirm that all assays showed superior performance discriminating mutant and wild-type alleles. Mutant alleles were successfully detected as low as 0.1%.
Gene expression profile of the tumor microenvironment from 40 NSCLC FFPE and ...Thermo Fisher Scientific
The tumor microenvironment (TME) is the intersection between tumor cells and
surrounding non-transformed cells. It contains immune cells, signaling molecules,
stromal and extracellular matrix. Research has shown the TME is often associated
with tumor growth. However, the function and regulatory mechanism of each
constituent is still poorly understood. The presence of PD-L1 is a promising marker
to predict positive response for T cell checkpoint therapy. Current IHC methods to
measure PD-L1 are subjective and highly variable. A higher-throughput and
standardized method that can systematically measure gene expression of cells
present in the TME has emerged to be a more desirable solution.
We applied the OncomineTM Immune Response Research Assay to measure the
expression of 395 genes in non-small cell lung cancer (NSCLC) research samples
from 40 matched FFPE and fresh frozen sample types. This assay covers genes
involved in checkpoint pathway, T cell regulation, cytokine and interferon signaling
pathways, and markers of different tumor infiltrating lymphocyte (TIL) subsets, as
well as tumor markers. With an input requirement of 10 ng of total RNA, libraries
were generated, templated on the Ion ChefTM and sequenced on the Ion S5TM
System. Sequencing data was analyzed and mapped with Torrent Suite Software
and differential expression analysis was conducted with AffymetrixTM Transcriptome
Analysis Console.
Detection and Surveillance of Antibiotic Resistance Genes From Food and Ferti...QIAGEN
One potential way to acquire antibiotic resistance genes is through the food supply chain. Both livestock and feed may
acquire antibiotic resistant bacteria via different mechanisms. Foodstuffs can be exposed to antibiotic resistant bacteria
through fertilizer originating from waste-water treatment plants. This, in addition to increasing administration of antibiotics
to livestock, can lead to food being a potential source of antibiotic resistance genes. This may lead to horizontal gene
transfer to pathogenic enteropathogens and further to drug resistance in humans. Therefore, the surveillance and prevention
of antibiotic resistance genes in food is important.
To effectively combat the spread of difficult-to-treat bacterial infections, rapid surveillance methods to detect antibiotic
resistance genes are required; in order to monitor both bacterial isolates and metagenomic samples.
Since the gut is known to act as a reservoir for antibiotic resistance genes, a small-scale research study was performed on
5 stool samples isolated from healthy human adults using an antibiotic resistance gene identification PCR array. In addition,
the diversity of antibiotic resistance genes in municipal biosolids was determined using an Antibiotic Resistance Genes
Microbial DNA qPCR Array with DNA extracted from belt-filter, press-cake sewage samples.
22 antibiotic resistance genes were identified from different resistance classifications. Further studies were performed in
beef, chicken, vegetable and pork samples. In conclusion, PCR arrays can be effective tools for detection of antibiotic
resistance genes from food samples and potential fertilizer sources.
This document summarizes tools and techniques for studying the innate immune system. It describes the differences between innate and adaptive immunity, the phases and cellular components of the innate response, pattern recognition receptors, signaling pathways, and technologies for research including PCR arrays and reporter assays. An example is given of using a PCR array to study the role of TLR3 in the response to Chlamydia infection, finding that TLR3 and IFN-β are major mediators. Another example shows using a reporter array to identify NF-κB and other pathways activated by cytokine stimulation.
The document discusses genetically modified (GM) crops and methods for detecting them. It begins with an introduction to GM crops, noting that they are plants modified using genetic engineering to introduce new traits, and that global GM crop acreage has increased significantly in recent decades. It then provides details on three main analytical approaches for detecting GM crops: detection to determine if a product is GM or not, identification of the specific GM crop or trait present, and quantification of GM content. Several DNA-based, protein-based, and trait-based methods are described, including polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), and lateral flow tests. The document compares the different methods and concludes with a discussion of the
Deeper Insight into Transcriptomes! Download the FlyerQIAGEN
Discover a new workflow for RT-PCR-based gene expression work
Accurate and biologically relevant results in RT-PCR-based
gene expression can be difficult to achieve. Successful
transcriptome work requires validated, reproducible targets
and high-quality technology. Recognizing the variability arising from sample physiology
and pathology, the influence of sample purification and
assay conditions, and the importance of access to easyto-
use software, QIAGEN experts developed a new gene
expression workflow. It will help you properly validate your
RT-PCR and gain the deepest insight into your result.
The document summarizes qBiomarker Somatic Mutation PCR Arrays, which are PCR-based assays that can rapidly and accurately detect somatic mutations in cancer samples. The arrays can detect mutations present at as little as 0.01% of DNA in a sample. They have been validated to work reliably with different sample types, including archived samples. The arrays cover a wide range of clinically relevant cancer mutations and allow screening of many mutations simultaneously in a single PCR run. The assays and content were selected based on published data on mutation frequencies and functional significance. The arrays provide a simple method for sensitive somatic mutation profiling to aid cancer research.
Extending miRQC’s dynamic range: amplifying the view of Limiting RNA samples ...QIAGEN
The original microRNA quality control (miRQC) study provided an in-depth analysis of commercially available microRNA (miRNA) quantification platforms. Specifically, twelve different
microarray, real-time PCR and small RNA sequencing platforms were assessed for reproducibility, sensitivity, accuracy, specificity and concordance of differential expression using a variety of sample types. Overall, each platform exhibited specific strengths and weaknesses, leading to the
final suggestion that a platform should be chosen on the basis of the experimental setting and the specific research questions. With this suggestion in mind, and the fact that liquid miRNA biopsies are an area of intense interest, we sought to expand the original miRQC study. For our “miRQC extension,” we benchmarked the QIAGEN miScript® PCR System with and without preamplification, and included a specific focus on routinely used biofluids. Concurrently, we benchmarked the miScript PCR System against another SYBR® Green miRNA detection platform. Overall, QIAGEN miScript demonstrated strong reproducibility and accuracy as well as superior detection rate and sensitivity in biofluids. Collectively, QIAGEN miScript provides the leading solution for novel miRNA discoveries.
Stratified Medicine in Cancer: The Role of HistopathologistDr. Shubhi Saxena
This document discusses stratified medicine approaches for cancer treatment. It describes how cellular pathologists classify gene mutations in cancer, including whether they are germline or somatic, synonymous or non-synonymous, activating or inactivating. Certain mutations can predict treatment response or resistance. Key driver mutations are discussed for lung cancer, including EGFR mutations and ALK translocations. EGFR mutant cancers may respond to EGFR tyrosine kinase inhibitors, while ALK rearrangements are targeted by crizotinib. Histopathology plays an important role in mutation detection and molecular testing to guide targeted therapies.
RNA profiling is a powerful technique for understanding cellular origins and disease states. Recent studies in a variety of diseases have revealed RNA signatures that are excellent biomarker candidates for understanding disease status and predicting progression.
Suppose you want to discover a biomarker. What are the major steps in discovering a biomarker when you start from a blood sample? Here is the story of a researcher who is trying to find blood-based biomarkers in autism spectrum disorders.
Toll-like Receptors in Inflammation: Host Defense Webinar Series Part 2QIAGEN
Toll-like receptors (TLRs) play an important role in the innate immune system and inflammation. TLRs recognize pathogen-associated molecular patterns and activate signaling pathways that induce inflammatory responses. This webinar discusses TLR signaling and the role of TLRs in inflammation, including their association with various diseases. It also explores how long non-coding RNAs and microRNAs help regulate TLR signaling pathways and the inflammatory response. The webinar promotes QIAGEN tools for studying TLRs, such as RT-PCR arrays, that can help profile gene and RNA expression changes related to TLR signaling.
- WPE-stem cells, a type of human prostate progenitor/stem cell, were initially highly sensitive to cadmium exposure, experiencing 90% cell loss after one week compared to 15% for a differentiated cell line. However, the stem cells quickly adapted and recovered their numbers within 4 weeks.
- During chronic cadmium exposure, the stem cells acquired characteristics of cancer cells like increased MMP-9 levels and colony formation along with decreased expression of tumor suppressors. They also showed signs of epithelial-mesenchymal transition.
- The initial sensitivity was linked to increased apoptosis factors while the later adaptation involved upregulation of cadmium resistance factors like metallothionein and downregulation of cadmium
miRNA profiling from blood challenges and recommendations - Download the articleQIAGEN
The discovery of stable miRNA species circulating in blood has led to increased research focus on disease-related variations in serum and plasma miRNA expression and the possibility that such variations could serve as noninvasive biomarkers for disease. Working with serum and plasma miRNA presents various challenges in purification and characterization. In this paper, we outline QIAGEN recommendations for robust purification and quantification, as well as reliable data normalization and analysis.
The document describes the development of two TaqMan digital PCR liquid biopsy assays to detect mutations in the TERT promoter region associated with cancer. The assays target the C228T and C250T mutations. Testing showed the assays could reliably detect the mutations at levels as low as 0.1% mutation frequency in genomic DNA samples. Assay design was challenging due to the high GC content and repetitive sequences in the TERT promoter region. Optimization of thermal cycling conditions was needed for accurate detection of the mutations by digital PCR.
Meeting the challenges of miRNA research: miRNA and its Role in Human Disease...QIAGEN
This document discusses a 4-part webinar series on microRNA (miRNA) research presented by QIAGEN. Part 1 will cover miRNA profiling from biofluids, part 2 will discuss challenges in miRNA research, part 3 will focus on advanced miRNA expression analysis, and part 4 will analyze functional analysis of miRNA. The document provides background on miRNAs and their role in gene expression and disease. It also describes QIAGEN products and solutions for miRNA sample preparation, real-time PCR, data analysis, and functional validation to help researchers overcome challenges in miRNA analysis.
Streamlined next generation sequencing assay development using a highly multi...Thermo Fisher Scientific
Next generation sequencing (NGS) assay development for solid tumor sequencing requires characterization of variant calling directly from formalin-fixed paraffin embedded (FFPE) tissue samples. However, cell line based FFPE and human FFPE samples only contain 2 to 20 variants, which require laboratories to invest significant resources in sample sourcing and preparation when developing assays to detect 100+ variants
PREDICTION OF ANTIMICROBIAL PEPTIDES USING MACHINE LEARNING METHODSBilal Nizami
Increasing resistance toward the conventional antibiotics has become a global concern. Antimicrobial peptides (AMPs) are potential alternatives for conventional antibiotics. Due to cost related reasons in designing and synthesis of AMPs. Machine learning based prediction tools are indispensable.
1) Researchers identified differential methylation of the GRIN2D gene in colon cancer cells using reduced representation bisulfite sequencing, finding it demethylated in approximately 60% of cell lines. 2) They confirmed this epigenetic alteration led to upregulated GRIN2D expression and assessed its role in the cancer phenotype. 3) Knockdown of GRIN2D expression in demethylated RKO colon cancer cells using shRNA corresponded to reduced cell growth, suggesting GRIN2D demethylation contributes to the tumor cell phenotype. Future replication of these findings is needed to fully understand the significance.
Antimicrobial peptides from Croaker FishAjit Antony
The document describes a study on antimicrobial peptides extracted from the croaker fish, Johnius dussumieri. The objective was to extract and characterize antimicrobial peptides from J. dussumieri. Peptides were extracted using acetic acid-acetone precipitation and tested against various gram-positive and gram-negative bacterial strains. Active fractions were purified using solid phase extraction and cation exchange chromatography. The purified fractions showed inhibition against test strains including Staphylococcus aureus, Bacillus cereus, and Vibrio alginolyticus. The study characterized antimicrobial peptides from J. dussumieri that have potential antimicrobial properties.
Maximizing PCR and RT-PCR Success - Download the BrochureQIAGEN
The invention of the polymerase chain reaction (PCR) by K. Mullis and coworkers in 1985 revolutionized molecular biology and molecular medicine. Major research areas, such as biomarker discovery, gene regulation and cancer research are challenging today’s PCR technologies with more demanding requirements. These include the need for increased throughput while reducing costs, higher assay sensitivity and reliable data normalization. Assay development and evaluation, reproducibility of data and time to result are still major problems encountered by researchers.
Meeting today’s challenges in PCR requires advances in all methods of the workflow that starts with sample collection, sample stabilization, and nucleic acid purification, and ends with amplification and detection. The following pages focus on the importance of amplification in meeting these challenges.
Detection and quantification of mutant alleles in tumor tissue allow for research disease monitoring and the research of drug efficacy. Detection of emerging secondary mutations in the same tumor tissue causing resistance to potential treatment will help guide decisions on future treatment plans. Testing for the presence of mutations in cell free DNA (cfDNA) is a less invasive research method than using tumor tissue. We created a research tool for mutation detection at a sensitivity level of 1% and below. This allows researchers to find correlation between types of mutations and types of tumors and determination of potential secondary mutations.
The tool combines TaqMan® SNP Genotyping Assays with digital PCR. A set of assays was optimized for use
in digital PCR with the QuantStudio® 3D Digital PCR System. In digital PCR, partitioning the sample into many individual reaction wells facilitates detection and quantification of rare mutant alleles. TaqMan® SNP Genotyping Assays ensure reliable discrimination of mutant and wild-type allele. Our current set of 60 assays covers mutations commonly found in tumor tissues, such as: BRAF V600E, mutations in EGFR exons 19, 20 and 21, KRAS codons 12 and 13, PIK3CA exons 9 and 20, and the JAK2 V617F mutations. All assays were wet-lab tested at a 10% mutation rate and a 1% mutation rate using mutant plasmid spiked into wild-type genomic DNA. Additionally, selected assays were tested at the 0.1% mutation rate using mutant cell lines spiked into wild-type genomic DNA. Wet-lab results confirm that all assays showed superior performance discriminating mutant and wild-type alleles. Mutant alleles were successfully detected as low as 0.1%.
Gene expression profile of the tumor microenvironment from 40 NSCLC FFPE and ...Thermo Fisher Scientific
The tumor microenvironment (TME) is the intersection between tumor cells and
surrounding non-transformed cells. It contains immune cells, signaling molecules,
stromal and extracellular matrix. Research has shown the TME is often associated
with tumor growth. However, the function and regulatory mechanism of each
constituent is still poorly understood. The presence of PD-L1 is a promising marker
to predict positive response for T cell checkpoint therapy. Current IHC methods to
measure PD-L1 are subjective and highly variable. A higher-throughput and
standardized method that can systematically measure gene expression of cells
present in the TME has emerged to be a more desirable solution.
We applied the OncomineTM Immune Response Research Assay to measure the
expression of 395 genes in non-small cell lung cancer (NSCLC) research samples
from 40 matched FFPE and fresh frozen sample types. This assay covers genes
involved in checkpoint pathway, T cell regulation, cytokine and interferon signaling
pathways, and markers of different tumor infiltrating lymphocyte (TIL) subsets, as
well as tumor markers. With an input requirement of 10 ng of total RNA, libraries
were generated, templated on the Ion ChefTM and sequenced on the Ion S5TM
System. Sequencing data was analyzed and mapped with Torrent Suite Software
and differential expression analysis was conducted with AffymetrixTM Transcriptome
Analysis Console.
The document discusses the FOXP2 protein and its effects on cortico-basal ganglia circuits, speech, and language. It describes how FOXP2 is encoded in humans and expressed in the basal ganglia and frontal cortex. Mutations can cause reduced speech and developmental issues. Mouse models with FOXP2 disruptions show motor impairments and early death. Two amino acid changes in human FOXP2 occurred recently in evolution and may have enabled complex vocal learning abilities unique to humans. The document also reviews brain regions where FOXP2 is expressed and disorders like apraxia that have been linked to it.
Thousands of different long non-coding RNAs (lncRNAs) exist in mammalian cells. lncRNAs do not encode proteins but can be very important for cell function. Studying their functions can be difficult because of their diverse modes of action. One method to discern cellular function is by selective knockdown of a specific lncRNA species. However, achieving consistent knockdown has proven to be more challenging for lncRNAs than for mRNAs or miRNAs. In this presentation, we discuss some of the issues encountered with lncRNA research. We cover antisense oligonucleotide (ASO) and small interfering RNA (siRNA) methods for lncRNA knockdown. And, we show how cellular localization of a specific lncRNA target informs the choice of knockdown method.
Knockout mice are mice that have had a specific gene deleted or inactivated through genetic engineering. This allows researchers to study the function of genes by observing the effects of the gene's absence. Researchers use embryonic stem cells from mice to delete genes through a process called homologous recombination. Mice with the gene deletion are bred to generate strains of mice lacking the gene. Studying these knockout mice provides insights into the roles and functions of genes. Knockout mice are also useful models for studying human diseases and evaluating potential treatments.
Gene knockout animals are genetically engineered organisms with inactivated genes. Mice are commonly used for knockout experiments due to their close genetic similarity to humans and low cost of breeding. Knocking out genes in mice provides insights into gene function and human disease modeling. The gene targeting method uses embryonic stem cells to precisely knockout a gene, while gene trapping does not require known gene sequences but confirmation of knockout is needed. Knockout studies have furthered understanding of cancer, obesity, and other diseases.
This document discusses microRNAs (miRNAs) and methods for studying their function and regulation of genes. It describes:
1) What miRNAs are, how they work by incorporating into the RISC complex and repressing target mRNAs through translational repression or degradation.
2) Techniques for manipulating miRNAs in cell lines using reporter assays, mimics, inhibitors and target protectors to study their effects on genes.
3) How to screen for miRNAs that regulate a target gene using ready-made cDNA panels and quantitative PCR. Several examples are provided of identifying miRNAs that regulate important cancer genes.
This document summarizes research modulating the protein TRIM21 to potentially treat autoimmune diseases like Sjögren's syndrome and systemic lupus erythematosus. Preliminary results found that certain drug compounds increased TRIM21's ability to reduce inflammatory responses. MicroRNA analysis identified differentially expressed miRNAs between disease patients and controls, targeting TRIM21, though further validation is needed. Overall, modulating TRIM21 and characterizing miRNA involvement shows promise for developing personalized miRNA-based therapies for autoimmune conditions.
History
Host pathogen interaction
R gene
Molecular techniques for detection of plant pathogens
Role of molecular techniques in resistance breeding Deployment of R genes and linked markers
Transgenic approaches in plant protection
Conclusion
Toll-like receptors (TLRs) play a key role in the innate immune response to pathogens and tissue damage by recognizing molecular patterns and initiating signaling pathways that induce inflammation. TLRs are expressed by immune cells like dendritic cells and activate these cells which then activate T cells and drive both innate and adaptive immune responses. MicroRNAs also regulate TLR signaling pathways and the inflammatory response by targeting mediators downstream of TLR activation. QIAGEN offers a variety of tools for studying TLR signaling and inflammation, including PCR arrays to profile gene and miRNA expression and assays for functional studies, protein analysis, and epigenetics.
This document summarizes a webinar series on microRNAs and their role in human disease. It introduces microRNA biogenesis, function, and analysis. The webinar series consists of three parts that will cover microRNA biogenesis and function, advanced microRNA expression analysis, and profiling microRNA expression in different sample types for biomarker development. The document provides an agenda for the first webinar that will discuss microRNA background, genomics, role in disease, isolation technologies, quantification technologies, profiling technologies, and functionalization technologies. It also advertises a newly released product.
This document discusses the interplay between Mycobacterium tuberculosis (Mtb) and the host immune environment, particularly in the context of HIV/TB co-infection. It presents several key findings:
1. Mtb can detect and respond to environmental cues within the macrophage such as oxidative stress, nutrient limitation, and changes in pH and chloride concentration. This allows Mtb to sense its intracellular location and immune status.
2. Reporter strains of Mtb show an accelerated transcriptional response to stresses like nitric oxide in vaccinated mice, indicating the immune response is developing faster.
3. Drugs like isoniazid have greater activity against intracellular Mtb in naive mice, suggesting the bacteria replicate more in this
This document describes a webinar on meeting the challenges of microRNA (miRNA) research. The webinar is part of a three-part series on miRNAs and their role in human disease. Webinar 1 focuses on an introduction to miRNA biogenesis, function, and analysis, and is presented by Jonathan Shaffer from QIAGEN. The webinar agenda covers topics on miRNA background, genomics, involvement in disease, isolation technologies, quantification technologies, profiling technologies, and functionalization technologies.
Unlocking the Potential of mRNA Vaccines and TherapeuticsMerck Life Sciences
Watch the presentation of this webinar here: https://bit.ly/3lNmkf7
The therapeutic potential of mRNA has been studied for decades and this exciting modality could potentially disrupt the biological market, in particular vaccine and novel therapies. This webinar will highlight the potential of mRNA therapies and focus on the manufacturing process's associated challenges, solutions and perspectives from synthesis to delivery.
mRNA has emerged as a promising modality for a wide range of therapeutics and vaccines and could become the break-through technology of this century. mRNA-based platform technologies could enable a more rapid response to infectious diseases, outbreaks or pandemics and allow efficient gene replacements or cancer treatments. mRNA represents a safer alternative to DNA-based therapies and the technology has recently advanced to overcome stability and efficacy challenges. Because of that, the industrialization of this technology is just in its infancy stages and bottlenecks exist around scalability, purity, and delivery which are key to establish and deliver the promise of such platform. This webinar will shed light on the potential of mRNA therapies and focus on the manufacturing process's associated challenges, solutions and perspectives from synthesis to delivery.
In this webinar, you will learn:
• The potential behind using mRNA as a therapeutic and vaccine
• The mRNA production process
• The challenges around mRNA production
• The solutions and perspectives for a robust manufacturing process
• mRNA delivery systems and their manufacturing
This document describes miScript miRNA PCR Arrays, which allow for the simultaneous detection of genome-wide or pathway-focused microRNA (miRNA) expression. It provides an overview of miRNA biology and research, details the miScript miRNA PCR Array system workflow from isolation to data analysis, and discusses applications in cancer research, development, differentiation, and genome-wide discovery. The system offers validated miRNA assays, controls, and optimized reagents to enable reproducible and reliable miRNA expression profiling from RNA samples.
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.
This study examined the role of miR-138-5p in regulating human melanoma cell proliferation. The study found that miR-138-5p expression was significantly higher in melanoma tissues compared to controls. Overexpression of miR-138-5p promoted proliferation of Me45 melanoma cells, while inhibition of miR-138-5p suppressed proliferation. Bioinformatics analysis predicted that miR-138-5p targets the 3'-UTR of hTERT, and luciferase assays confirmed this. Knockdown of hTERT reversed the promotive effects of miR-138-5p on cell proliferation, indicating miR-138-5p regulates proliferation by directly targeting hTERT. Therefore, this study demonstrates that miR-138-5
Cisplatin (DDP) is a widely used chemotherapy drug for advanced cervical
cancer (CC), but resistance poses a significant challenge. While miR-4739 has been
implicated in tumor development, its specific role in regulating DDP resistance in CC
remains unclear.
Robert Anders, MD, PhD, Julie R. Brahmer, MD, MSc, and Christopher D. Gocke, MD, prepared useful Practice Aids pertaining to immunotherapy and biomarker testing for this CME/MOC/CC activity titled "Keeping Up With Advances in Cancer Immunotherapy and Biomarker Testing: Implications for Pathologists at the Forefront of the Emerging Precision Immuno-Oncology Era." For the full presentation, monograph, complete CME/MOC/CC information, and to apply for credit, please visit us at http://bit.ly/2L7zlSy. CME/MOC/CC credit will be available until May 2, 2020.
The document discusses the role of inflammation in cancer promotion. It describes an experiment using Mdr2-KO mice, which develop hepatocellular carcinoma in the context of chronic hepatitis. Nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) activation is involved in hepatocarcinogenesis in these mice and is predominantly located near inflamed portal tracts. Treatment with anti-inflammatory drugs or anti-TNFα decreases inflammation and NFkB activation in these mice. Generating double mutant mice with NFkB inactivated in hepatocytes showed that NFkB is critical for tumor promotion but dispensable for early stages of hepatocellular carcinoma development.
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.
This document discusses genomic markers for studying parasitic infections. It begins with an introduction on the challenges of studying parasites and how genomic tools can help overcome these challenges. It then describes several techniques for detecting and analyzing parasite genes, including PCR, real-time PCR, LAMP, LCR, Luminex, FISH, microarrays, and biosensors. The document also discusses tools for molecular epidemiology of parasites such as MLEE, AFLP, RAPD, PCR-RFLP, and MLMT. Finally, it provides examples of how these various genomic tools have been applied to study different parasite species.
This document describes a study that conducted an RNA interference screening in breast cancer cells to identify epigenetic factors regulating the mesenchyme to epithelium transition (MET). Researchers designed a siRNA library targeting 729 chromatin modification genes and screened it in the mesenchymal breast cancer cell line MDA-MB-231. They identified 70 candidate genes involved in MET, including known genes like ZEB1, G9a, SMAD5 and SMARCD3, as well as DOT1L which has been implicated in MET. They also identified KAT5 as a novel gene linked to maintaining the mesenchymal phenotype for the first time. The screening approach involved measuring E-cadherin induction and cell
Dr. Laura Miller - Comparative analysis of signature genes in PRRSV-infected ...John Blue
Comparative analysis of signature genes in PRRSV-infected porcine monocyte-derived dendritic cells at differential activation statuses - Dr. Laura Miller, Virus and Prion Diseases of Livestock Research Unit, National Animal Disease Center, USDA-ARS, from the 2015 North American PRRS Symposium, December 4 - 5, 2015, Chicago, IL, USA.
More presentations at http://www.swinecast.com/2015-north-american-prrs-symposium
Detection of rare mutations in tumor tissue and cell free DNA (cfDNA) allows for monitoring of tumor progression and regression for research purposes. cfDNA isolated from plasma combined with a sensitive detection method like digital PCR is non- invasive and enables earlier detection compared to conventional imaging techniques. Building on the TaqMan based Rare Mutation assay set for detection of rare mutations using digital PCR on the QuantStudio 3D Digital PCR System, we are now developing multiplex assays for simultaneous detection of several mutations. We selected relevant mutations in the EGFR and KRAS genes for our initial multiplex application: EGFR G719, EGFR exon 19 deletions, and
KRAS G12/G13. These mutations may have implications for potential future targeted therapy. Primers and probes of singleplex Rare Mutation Assays were reformulated to generate multiplex assays detecting the EGFR and KRAS mutations. All multiplex assays were tested on template composed of wild-type genomic DNA background mixed with mutant plasmid reflecting each of the mutations detected by the multiplex
assays. Initial experimental results were successful and showed excellent signal intensity and clear cluster separation when analyzed with the QuantStudio 3D AnalysisSuiteTM Cloud Software. The EGFR G719 mutations (COSM6239, COSM6253, COSM6252) were detected using a 3plex assay, EGFR exon 19 deletions (COSM12383, COSM12422, COSM12678, COSM6223, COSM6254, COSM6255) were detected using a 6plex assay, and KRAS G12/G13 mutations (COSM516,
COSM517, COSM518, COSM520, COSM521, COSM522, COSM527, COSM532) were detected using an 8plex. Multiplexing assays for three relevant mutation loci proved feasible and presents an efficient way to assess the presence and the percentage of mutations at these loci.
This slide is about the basics of mRNA-based therapy. The content includes: definition of mRNA, timeline of mRNA therapeutics, action mechanism and development strategies of mRNA drugs, therapeutic mRNA applications, and the related services provided by Creative Biolabs.
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
3. Introduction: Inflammation
̣
Definition: a protective tissue response to tissue damage or microbes, which serves to
destroy, dilute, or wall off both the injurious agent and the injured tissues.
Epigenetic
Changes
Microbes/Infection
Tissue Damage
Acute Inflammation
Infection Clearance
Tissue Homeostasis
mRNA
Changes
Cytokines & Chemokines
Signaling Pathways
Immune system composition
Chronic Inflammation
Pre-cancer & Cancer
Chronic Inflammatory Diseases
-3-
Sample & Assay Technologies
5. Basic principles of qRT-PCR: Overview
Real-Time PCR
Amplify and simultaneously quantify target DNA
Reverse Transcription Real-Time PCR
Amplify and simultaneously quantify mRNA
For more information and webinars on real-time PCR, visit:
www.sabiosciences.com/seminarlist.php
-5-
Sample & Assay Technologies
6. Experimental overview: Gene expression
analysis
Isolate RNA
Stimulate Cells
RT-PCR Arrays or Assays
Isolate DNA
Data Analysis
-6-
Sample & Assay Technologies
7. Gene expression in Inflammation
̣
Definition: a protective tissue response to tissue damage or microbes, which serves to
destroy, dilute, or wall off both the injurious agent and the injured tissues.
Epigenetic
Changes
Microbes/Infection
Tissue Damage
Acute Inflammation
Infection Clearance
Tissue Homeostasis
mRNA
Changes
Cytokines & Chemokines
Signaling Pathways
Immune system composition
Chronic Inflammation
Pre-cancer & Cancer
Chronic Inflammatory Diseases
-7-
Sample & Assay Technologies
8. ̣
Inflammation studies: Gene expression
The transcription factor cMyb is upregulated in naïve CD4 T cells.
-8-
Sample & Assay Technologies
9. ̣
Inflammation studies: Gene expression
The transcription factor cMyb is upregulated in naïve CD4 T cells.
-9-
Sample & Assay Technologies
10. ̣
Inflammation studies: Gene expression
The transcription factor cMyb is upregulated in naïve CD4 T cells. What is the effect of cMyb
on naïve CD4 T cell maturation (to Th1, Th2, or Th3)?
- 10 -
Sample & Assay Technologies
11. Inflammation studies: Gene expression
The transcription factor cMyb is upregulated in naïve CD4 T cells. What is the effect of cMyb
on naïve CD4 T cell maturation (to Th1, Th2, or Th3)?
̣
Experiment: Knockdown cMyb in human peripheral CD4+ T cells. Examine gene changes
using the Human Th1-Th2-Th3 RT2 Profiler PCR Array (PAHS-034).
̣
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Sample & Assay Technologies
12. Inflammation studies: Gene expression
The transcription factor cMyb is upregulated in naïve CD4 T cells. What is the effect of cMyb
on naïve CD4 T cell maturation (to Th1, Th2, or Th3)?
̣
Experiment: Knockdown cMyb in human peripheral CD4+ T cells. Examine gene changes
using the Human Th1-Th2-Th3 RT2 Profiler PCR Array (PAHS-034).
̣
Results:
̣
- 12 -
Sample & Assay Technologies
13. Inflammation studies: Gene expression
The transcription factor cMyb is upregulated in naïve CD4 T cells. What is the effect of cMyb
on naïve CD4 T cell maturation (to Th1, Th2, or Th3)?
̣
Experiment: Knockdown cMyb in human peripheral CD4+ T cells. Examine gene changes
using the Human Th1-Th2-Th3 RT2 Profiler PCR Array (PAHS-034).
̣
Results:
̣
Conclusions: Knockdown of cMyb in human peripheral CD4+ T cells decreased the
expression of Th2 cytokine genes, and negatively affected Th2 cell maturation in primary
human peripheral blood T cells.
̣
- 13 -
Sample & Assay Technologies
14. Gene expression in Inflammation
̣
Definition: a protective tissue response to tissue damage or microbes, which serves to
destroy, dilute, or wall off both the injurious agent and the injured tissues.
Epigenetic
Changes
Microbes/Infection
Tissue Damage
Acute Inflammation
Infection Clearance
Tissue Homeostasis
mRNA
Changes
Cytokines & Chemokines
Signaling Pathways
Immune system composition
Chronic Inflammation
Pre-cancer & Cancer
Chronic Inflammatory Diseases
- 14 -
Sample & Assay Technologies
15. ̣
Inflammation studies: Gene expression
Limited material
Mucin depleted foci (MDF) are precancerous lesions of the colon that show signs of
inflammation.
- 15 -
Sample & Assay Technologies
16. ̣
Inflammation studies: Gene expression
Limited material
Mucin depleted foci (MDF) are precancerous lesions of the colon that show signs of
inflammation.
- 16 -
Sample & Assay Technologies
17. ̣
Inflammation studies: Gene expression
Limited material
Mucin depleted foci (MDF) are precancerous lesions of the colon that show signs of
inflammation. What roles do TLRs play in MDF?
- 17 -
Sample & Assay Technologies
18. Inflammation studies: Gene expression
Limited material
Mucin depleted foci (MDF) are precancerous lesions of the colon that show signs of
inflammation. What roles do TLRs play in MDF?
̣
Experiment: Treat mice with DMH for 15 weeks to induce MDF. Collect RNA from MDF and
normal colon. Convert mRNA to cDNA. Pre-amplify cDNA with the Mouse TLR PreAMP
primer mixes (PBM-0018). Analyze differential gene expression with the Mouse TLR RT2
Profiler PCR Arrays (PAMM-018)
̣
- 18 -
Sample & Assay Technologies
19. Inflammation studies: Gene expression
Limited material
Mucin depleted foci (MDF) are precancerous lesions of the colon that show signs of
inflammation. What roles do TLRs play in MDF?
̣
Experiment: Treat mice with DMH for 15 weeks to induce MDF. Collect RNA from MDF and
normal colon. Pre-amplify cDNA with the Mouse TLR PreAMP primer mixes (PBM-0018).
Analyze differential gene expression with the Mouse TLR RT2 Profiler PCR Arrays (PAMM018)
̣
Results:
̣
- 19 -
Sample & Assay Technologies
20. Inflammation studies: Gene expression
Limited material
Mucin depleted foci (MDF) are precancerous lesions of the colon that show signs of
inflammation. What roles do TLRs play in MDF?
̣
Experiment: Treat mice with DMH for 15 weeks to induce MDF. Collect RNA from MDF and
normal colon. Pre-amplify cDNA with the Mouse TLR PreAMP primer mixes (PBM-0018).
Analyze differential gene expression with the Mouse TLR RT2 Profiler PCR Arrays (PAMM018)
̣
Results:
̣
Conclusion: MDF induces TLR2 gene expression in MDF compared to normal colon,
suggesting a link between TLR2 and MDF.
̣
- 20 -
Sample & Assay Technologies
22. Epigenetics: Overview
Activated
Transcription Factors
miRNA
shRNA
siRNA
Protein “A”
NFκB
+
p53
Transcription
Initiation Complex
mRNA ”A”
–
Histones
p53 BS Me
Me
Me
Me Me
NFκB BS
DNA Methylation
Histone-DNA
Interactions
Ac
Structural Gene
Me
Me Me
DNA Methylation
For more information and webinars on Epigenetics, visit:
www.sabiosciences.com/seminarlist.php
- 22 -
Sample & Assay Technologies
24. miRNA in Inflammation
̣
Definition: a protective tissue response to tissue damage or microbes, which serves to
destroy, dilute, or wall off both the injurious agent and the injured tissues.
Microbes/Infection
Tissue Damage
Epigenetic
Changes
(miRNA)
mRNA
Changes
Acute Inflammation
Infection Clearance
Tissue Homeostasis
Cytokines & Chemokines
Signaling Pathways
Immune system composition
Chronic Inflammation
Pre-cancer & Cancer
Chronic Inflammatory Diseases
- 24 -
Sample & Assay Technologies
25. ̣
Inflammation studies: miRNA function
Resolution of inflammation is a tightly controlled mechanism. The D-series resolvins (RvD1)
are a class of players that mediate this resolution.
- 25 -
Sample & Assay Technologies
26. ̣
Inflammation studies: miRNA function
Resolution of inflammation is a tightly controlled mechanism. The D-series resolvins (RvD1)
are a class of players that mediate this resolution.
FASEB J. 25, 1–17 (2011)
- 26 -
Sample & Assay Technologies
27. ̣
Inflammation studies: miRNA function
Resolution of inflammation is a tightly controlled mechanism. The D-series resolvins (RvD1)
are a class of players that mediate this resolution. Can RvD1-mediated resolution happen
through miRNAs?
FASEB J. 25, 1–17 (2011)
- 27 -
Sample & Assay Technologies
28. Inflammation studies: miRNA function
Resolution of inflammation is a tightly controlled mechanism. The D-series resolvins (RvD1)
are a class of players that mediate this resolution. Can RvD1-mediated resolution happen
through miRNAs?
̣
̣
Experiment: Treat mice with zymosan A to induce acute inflammation (murine peritonitis), in
the presence or absence of RvD1, for 4, 12, or 24 hours (representing onset, maximum,
and resolution phases). Collect RNA from leukocytes. Analyze miRNA expression with the
Mouse miRNome miRNA PCR Array (MIMM-216Z)
FASEB J. 25, 1–17 (2011)
- 28 -
Sample & Assay Technologies
29. Inflammation studies: miRNA function
Resolution of inflammation is a tightly controlled mechanism. The D-series resolvins (RvD1)
are a class of players that mediate this resolution. Can RvD1-mediated resolution happen
through miRNAs?
̣
Experiment: Treat mice with zymosan A to induce acute inflammation (murine peritonitis), in
the presence or absence of RvD1, for 4, 12, or 24 hours (representing onset, maximum,
and resolution phases). Collect RNA from leukocytes. Analyze miRNA expression with the
Mouse miRNome miRNA PCR Array (MIMM-216Z)
̣
̣
Results:
- 29 -
Sample & Assay Technologies
30. Inflammation studies: miRNA function
Resolution of inflammation is a tightly controlled mechanism. The D-series resolvins (RvD1)
are a class of players that mediate this resolution. Can RvD1-mediated resolution happen
through miRNAs?
̣
Experiment: Treat mice with zymosan A to induce acute inflammation (murine peritonitis), in
the presence or absence of RvD1, for 4, 12, or 24 hours (representing onset, maximum,
and resolution phases). Collect RNA from leukocytes. Analyze miRNA expression with the
Mouse miRNome miRNA PCR Array (MIMM-216Z)
̣
Results:
̣
̣
Conclusion: RvD1 regulated resolution by controlling the expression of miR-219 and miR146b. These miRNAs are the first identified miRNAs in resolvin resolution circuits.
- 30 -
Sample & Assay Technologies
32. DNA methylation in Inflammation
̣
Definition: a protective tissue response to tissue damage or microbes, which serves to
destroy, dilute, or wall off both the injurious agent and the injured tissues.
Microbes/Infection
Tissue Damage
Epigenetic
Changes
(DNA Methylation)
mRNA
Changes
Acute Inflammation
Infection Clearance
Tissue Homeostasis
Cytokines & Chemokines
Signaling Pathways
Immune system composition
Chronic Inflammation
Pre-cancer & Cancer
Chronic Inflammatory Diseases
- 32 -
Sample & Assay Technologies
33. Inflammation studies: DNA methylation
ANCA Vasculitis is characterized by microvascular inflammation caused by activated
neutrophils. These activated neutrophils aberrantly express PR3 and MPO, which are
silenced in normal individuals.
- 33 -
Sample & Assay Technologies
34. Inflammation studies: DNA methylation
ANCA Vasculitis is characterized by microvascular inflammation caused by activated
neutrophils. These activated neutrophils aberrantly express PR3 and MPO, which are
silenced in normal individuals.
- 34 -
Sample & Assay Technologies
35. Inflammation studies: DNA methylation
ANCA Vasculitis is characterized by microvascular inflammation caused by activated
neutrophils. These activated neutrophils aberrantly express PR3 and MPO, which are
silenced in normal individuals.
Does aberrant PR3 and MPO expression result from disrupted epigenetic silencing?
- 35 -
Sample & Assay Technologies
36. Inflammation studies: DNA methylation
ANCA Vasculitis is characterized by microvascular inflammation caused by activated
neutrophils. These activated neutrophils aberrantly express PR3 and MPO, which are
silenced in normal individuals.
Does aberrant PR3 and MPO expression result from disrupted epigenetic silencing?
Experiment: Isolate gDNA from neutrophils and treat with methylation-sensitive and
methylation-dependent restriction enzymes (EpiTect Methyl DNA Restriction Kit 335451).
Analyze DNA methylation by real-time PCR using primers specific for CpG islands in PR3
(MePH23428-1A) and MPO (MePH22382-1A).
- 36 -
Sample & Assay Technologies
37. Inflammation studies: DNA methylation
ANCA Vasculitis is characterized by microvascular inflammation caused by activated
neutrophils. These activated neutrophils aberrantly express PR3 and MPO, which are
silenced in normal individuals.
Does aberrant PR3 and MPO expression result from disrupted epigenetic silencing?
Experiment: Isolate gDNA from neutrophils and treat with methylation-sensitive and
methylation-dependent restriction enzymes (EpiTect Methyl DNA Restriction Kit 335451).
Analyze DNA methylation by real-time PCR using primers specific for CpG islands in PR3
(MePH23428-1A) and MPO (MePH22382-1A).
Results:
- 37 -
Sample & Assay Technologies
38. Inflammation studies: DNA methylation
ANCA Vasculitis is characterized by microvascular inflammation caused by activated
neutrophils. These activated neutrophils aberrantly express PR3 and MPO, which are
silenced in normal individuals.
Does aberrant PR3 and MPO expression result from disrupted epigenetic silencing?
Experiment: Isolate gDNA from neutrophils and treat with methylation-sensitive and
methylation-dependent restriction enzymes (EpiTect Methyl DNA Restriction Kit 335451).
Analyze DNA methylation by real-time PCR using primers specific for CpG islands in PR3
(MePH23428-1A) and MPO (MePH22382-1A).
Results:
Conclusion: Methylation of CpG islands is a mechanism that controls the expression of MPO,
but not the expression of PR3.
The EpiTect Methyl System uses the MethylScreen™
Technology provided under license from Orion Genomics, LLC.
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Sample & Assay Technologies
40. Histone modification in Inflammation
̣
Definition: a protective tissue response to tissue damage or microbes, which serves to
destroy, dilute, or wall off both the injurious agent and the injured tissues.
Microbes/Infection
Tissue Damage
Epigenetic
Changes
(Histone Modifications)
mRNA
Changes
Acute Inflammation
Infection Clearance
Tissue Homeostasis
Cytokines & Chemokines
Signaling Pathways
Immune system composition
Chronic Inflammation
Pre-cancer & Cancer
Chronic Inflammatory Diseases
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Sample & Assay Technologies
41. Inflammation studies: Histone modification
Th17 cells are a subset of CD4 T cells that differentiate from naïve T cells, secrete IL-17, and
induce massive tissue inflammation. T cell factor 1 (TCF-1) is a transcription factor that plays
an important role in T cell differentiation in the periphery.
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Sample & Assay Technologies
42. Inflammation studies: Histone modification
Th17 cells are a subset of CD4 T cells that differentiate from naïve T cells, secrete IL-17, and
induce massive tissue inflammation. T cell factor 1 (TCF-1) is a transcription factor that plays
an important role in T cell differentiation in the periphery.
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Sample & Assay Technologies
43. Inflammation studies: Histone modification
Th17 cells are a subset of CD4 T cells that differentiate from naïve T cells, secrete IL-17, and
induce massive tissue inflammation. T cell factor 1 (TCF-1) is a transcription factor that plays
an important role in T cell differentiation in the periphery. Does TCF-1 control the
differentiation of Th17 cells, and how?
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Sample & Assay Technologies
44. Inflammation studies: Histone modification
Th17 cells are a subset of CD4 T cells that differentiate from naïve T cells, secrete IL-17, and
induce massive tissue inflammation. T cell factor 1 (TCF-1) is a transcription factor that plays
an important role in T cell differentiation in the periphery. Does TCF-1 control the
differentiation of Th17 cells, and how?
Experiment: Isolate naïve T cells from spleens of mice. Induce Th17 cell differentiation.
Collect chromatin and process with the EpiTect ChIP one-day kit (334471) for chromatin
immunoprecipitation with anti-histone antibodies. Detect precipitated DNA with qRT-PCR.
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Sample & Assay Technologies
45. Inflammation studies: Histone modification
Th17 cells are a subset of CD4 T cells that differentiate from naïve T cells, secrete IL-17, and
induce massive tissue inflammation. T cell factor 1 (TCF-1) is a transcription factor that plays
an important role in T cell differentiation in the periphery. Does TCF-1 control the
differentiation of Th17 cells, and how?
Experiment: Isolate naïve T cells from spleens of mice. Induce aTh17 cell differentiation.
Collect chromatin and process with the EpiTect ChIP one-day kit for chromatin
immunoprecipitation with anti-histone antibodies. Detect precipitated DNA with qRT-PCR.
Results:
Open chromatin = increased expression
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Sample & Assay Technologies
46. Inflammation studies: Histone modification
Th17 cells are a subset of CD4 T cells that differentiate from naïve T cells, secrete IL-17, and
induce massive tissue inflammation. T cell factor 1 (TCF-1) is a transcription factor that plays
an important role in T cell differentiation in the periphery. Does TCF-1 control the
differentiation of Th17 cells, and how?
Experiment: Isolate naïve T cells from spleens of mice. Induce Th17 cell differentiation.
Collect chromatin and process with the EpiTect ChIP one-day kit (334471) for chromatin
immunoprecipitation with anti-histone antibodies. Detect precipitated DNA with qRT-PCR.
Results:
Conclusion: TCF-1 mediates the repression of IL-17 locus during T cell development by
chromatin modifications.
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Sample & Assay Technologies
48. Cignal Reporter Assays: Complete Solution
Reporter assays: Overview
Transcriptional Regulatory Elements (TRE), which establish the
specificity of each reporter
TATA
box
Reporter Construct
GFP/firefly luciferase
Tandem repeats of
TRE
EGFP
TF
FL
Upstream Signaling
Events
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Sample & Assay Technologies
49. Reporter assays for Inflammation
̣
Definition: a protective tissue response to tissue damage or microbes, which serves to
destroy, dilute, or wall off both the injurious agent and the injured tissues.
Epigenetic
Changes
Microbes/Infection
Tissue Damage
Acute Inflammation
Infection Clearance
Tissue Homeostasis
mRNA
Changes
Cytokines & Chemokines
Signaling Pathways
Immune system composition
Chronic Inflammation
Pre-cancer & Cancer
Chronic Inflammatory Diseases
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Sample & Assay Technologies
50. ̣
Inflammation studies: Reporter assay
Neisseria Meningitidis infections can be rapidly fatal due to acute inflammatory responses
that are mediated by capsular polysaccharides (CPS), but the innate immunostimulatory
activity of CPS is largely unknown.
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Sample & Assay Technologies
51. ̣
Inflammation studies: Reporter assay
Neisseria Meningitidis infections can be rapidly fatal due to acute inflammatory responses
that are mediated by capsular polysaccharides (CPS), but the innate immunostimulatory
activity of CPS is largely unknown.
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Sample & Assay Technologies
52. ̣
Inflammation studies: Reporter assay
Neisseria Meningitidis infections can be rapidly fatal due to acute inflammatory responses
that are mediated by capsular polysaccharides (CPS), but the innate immunostimulatory
activity of CPS is largely unknown. What signaling pathways are induced upon CPS
recognition?
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Sample & Assay Technologies
53. Inflammation studies: Reporter assay
Neisseria Meningitidis infections can be rapidly fatal due to acute inflammatory responses
that are mediated by capsular polysaccharides (CPS), but the innate immunostimulatory
activity of CPS is largely unknown. What signaling pathways are induced upon CPS
recognition?
̣
Experiment: Reverse-transfect cells with 10 dual-luciferase reporter assays, individually, on a
reporter array (Cignal Finder 10-pathway Reporter Array; CCA-108L). Induce transfected
cells with CPS, and measure luciferase levels to determine which pathway(s) is(are) activated
by CPS
̣
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Sample & Assay Technologies
54. Inflammation studies: Reporter assay
Neisseria Meningitidis infections can be rapidly fatal due to acute inflammatory responses
that are mediated by capsular polysaccharides (CPS), but the innate immunostimulatory
activity of CPS is largely unknown. What signaling pathways are induced upon CPS
recognition?
̣
Experiment: Reverse-transfect cells with 10 dual-luciferase reporter assays, individually, on a
reporter array (Cignal Finder 10-pathway Reporter Array; CCA-108L). Induce transfected
cells with CPS, and measure luciferase levels to determine which pathway(s) is(are) activated
by CPS
̣
Results:
̣
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Sample & Assay Technologies
55. Inflammation studies: Reporter assay
Neisseria Meningitidis infections can be rapidly fatal due to acute inflammatory responses
that are mediated by capsular polysaccharides (CPS), but the innate immunostimulatory
activity of CPS is largely unknown. What signaling pathways are induced upon CPS
recognition?
̣
Experiment: Reverse-transfect cells with 10 dual-luciferase reporter assays, individually, on a
reporter array (Cignal Finder 10-pathway Reporter Array; CCA-108L). Induce transfected
cells with CPS, and measure luciferase levels to determine which pathway(s) is(are) activated
by CPS
̣
Results:
̣
Conclusions: NFkB is the major signaling pathway activated in response to CPS.
̣
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Sample & Assay Technologies
57. Gene knockdown for Inflammation
̣
Definition: a protective tissue response to tissue damage or microbes, which serves to
destroy, dilute, or wall off both the injurious agent and the injured tissues.
Epigenetic
Changes
Microbes/Infection
Tissue Damage
Acute Inflammation
Infection Clearance
Tissue Homeostasis
mRNA
Changes
Cytokines & Chemokines
Signaling Pathways
Immune system composition
Chronic Inflammation
Pre-cancer & Cancer
Chronic Inflammatory Diseases
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Sample & Assay Technologies
58. ̣
Inflammation studies: siRNA knockdown
Resident alveolar macrophages (AM) constitute the first line of defense against invading lung
Streptococcus pneumoniae. Studies have shown that morphine-treated mice experience
increased mortality and bacterial outgrowth and dissemination.
- 58 -
Sample & Assay Technologies
59. ̣
Inflammation studies: siRNA knockdown
Resident alveolar macrophages (AM) constitute the first line of defense against invading lung
Streptococcus pneumoniae. Studies have shown that morphine-treated mice experience
increased mortality and bacterial outgrowth and dissemination.
The Journal of Immunology, 2008, 180: 3594–3600.
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Sample & Assay Technologies
60. ̣
Inflammation studies: siRNA knockdown
Resident alveolar macrophages (AM) constitute the first line of defense against invading lung
Streptococcus pneumoniae. Studies have shown that morphine-treated mice experience
increased mortality and bacterial outgrowth and dissemination. What is the mechanism by
which morphine does this?
The Journal of Immunology, 2008, 180: 3594–3600.
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Sample & Assay Technologies
61. Inflammation studies: siRNA knockdown
Resident alveolar macrophages (AM) constitute the first line of defense against invading lung
Streptococcus pneumoniae. Studies have shown that morphine-treated mice experience
increased mortality and bacterial outgrowth and dissemination. What is the mechanism by
which morphine does this?
̣
Experiment: MH-S cells (murine AM cells) were transfected with either a negative control
SureSilencing shRNA or a SureSilencing shRNA plasmid for mouse TLR9. Levels of MIP-2
were measured after treating transfected cells with morphine and Streptococcus pneumoniae
̣
The Journal of Immunology, 2008, 180: 3594–3600.
- 61 -
Sample & Assay Technologies
62. Inflammation studies: siRNA knockdown
Resident alveolar macrophages (AM) constitute the first line of defense against invading lung
Streptococcus pneumoniae. Studies have shown that morphine-treated mice experience
increased mortality and bacterial outgrowth and dissemination. What is the mechanism by
which morphine does this?
̣
Experiment: MH-S cells (murine AM cells) were transfected with either a negative control
SureSilencing shRNA or a SureSilencing shRNA plasmid for mouse TLR9. Levels of MIP-2
were measured after treating transfected cells with morphine and Streptococcus pneumoniae.
̣
Results:
̣
- 62 -
Sample & Assay Technologies
63. Inflammation studies: siRNA knockdown
Resident alveolar macrophages (AM) constitute the first line of defense against invading lung
Streptococcus pneumoniae. Studies have shown that morphine-treated mice experience
increased mortality and bacterial outgrowth and dissemination. What is the mechanism by
which morphine does this?
̣
Experiment: MH-S cells (murine AM cells) were transfected with either a negative control
SureSilencing shRNA or a SureSilencing shRNA plasmid for mouse TLR9. Levels of MIP-2
were measured after treating transfected cells with morphine and Streptococcus pneumoniae.
̣
Results:
̣
Conclusion: Morphine reduces levels of Streptococcus pneumoniae -induced MIP-2 from AM
cells in a TLR9-dependent manner.
̣
- 63 -
Sample & Assay Technologies
65. Chemokine expression in Inflammation
̣
Definition: a protective tissue response to tissue damage or microbes, which serves to
destroy, dilute, or wall off both the injurious agent and the injured tissues.
Epigenetic
Changes
Microbes/Infection
Tissue Damage
Acute Inflammation
Infection Clearance
Tissue Homeostasis
mRNA
Changes
Cytokines & Chemokines
Signaling Pathways
Immune system composition
Chronic Inflammation
Pre-cancer & Cancer
Chronic Inflammatory Diseases
- 65 -
Sample & Assay Technologies
66. ̣
Inflammation studies: Chemokine levels
Diabetes mellitus (DM) is a major risk factor for tuberculosis (TB) but the defect in protective
immunity responsible for this has not been defined.
- 66 -
Sample & Assay Technologies
67. ̣
Inflammation studies: Chemokine levels
Diabetes mellitus (DM) is a major risk factor for tuberculosis (TB) but the defect in protective
immunity responsible for this has not been defined.
The Journal of Immunology, 2010, 184: 6275–6282
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Sample & Assay Technologies
68. ̣
Inflammation studies: Chemokine levels
Diabetes mellitus (DM) is a major risk factor for tuberculosis (TB) but the defect in protective
immunity responsible for this has not been defined. Could this be due to defective chemokine
secretion leading to delayed priming of adaptive immunity?
The Journal of Immunology, 2010, 184: 6275–6282
- 68 -
Sample & Assay Technologies
69. Inflammation studies: Chemokine levels
Diabetes mellitus (DM) is a major risk factor for tuberculosis (TB) but the defect in protective
immunity responsible for this has not been defined. Could this be due to defective chemokine
secretion leading to delayed priming of adaptive immunity?
̣
Experiment: Lung homogenates from TB-infected Diabetic mice were tested for chemokine
levels using the Multi-Analyte ELISArray Kit.
̣
The Journal of Immunology, 2010, 184: 6275–6282
- 69 -
Sample & Assay Technologies
70. Inflammation studies: Chemokine levels
Diabetes mellitus (DM) is a major risk factor for tuberculosis (TB) but the defect in protective
immunity responsible for this has not been defined. Could this be due to defective chemokine
secretion leading to delayed priming of adaptive immunity?
̣
Experiment: Lung homogenates from TB-infected Diabetic mice were tested for chemokine
levels using the ELISArray.
̣
Results:
̣
- 70 -
Sample & Assay Technologies
71. Inflammation studies: Chemokine levels
Diabetes mellitus (DM) is a major risk factor for tuberculosis (TB) but the defect in protective
immunity responsible for this has not been defined. Could this be due to defective chemokine
secretion leading to delayed priming of adaptive immunity?
̣
Experiment: Lung homogenates from TB-infected Diabetic mice were tested for chemokine
levels using the Multi-Analyte ELISArray Kit.
̣
Results:
̣
Conclusion: Reduced levels of leukocyte chemoattractant factors including CCL2 and CCL5
at early timepoints post-infection could explain why diabetic mice are more prone to TB.
̣
- 71 -
Sample & Assay Technologies
72. Conclusions
QIAGEN offers many methods to study
molecular and cellular mechanisms involved in
Inflammation:
Gene Expression
RT2 Profiler PCR Arrays & Assays
RT2 PreAMP Primer Mixes
Epigenetics
miScript miRNA PCR System
EpiTect Methyl qPCR Arrays
EpiTect ChIP qPCR Arrays
Functional Studies
Cignal Reporter Assays
SureSilencing shRNA Plasmid
Protein expression
ELISArray
̣
www.sabiosciences.com
̣
- 72 -
Sample & Assay Technologies
73. Keep up to date: Follow Pathway focused biology on Facebook
Latest
information on
pathway and
disease
research,
resources and
demos.
- 73 -
Sample & Assay Technologies
74. Thank you for attending!
Would you like to try an Inflammation-related PCR Array?
RT2 Profiler PCR Array Starter Pack
miScript PCR Array Starter Pack
PCR Arrays of any Pathway (FREE)
• 2 96-well/100-well (2 samples) OR
• 1 384-well (4 samples)
• Required Reagents (w/ Purchase)
• RT2 First-Strand cDNA Synthesis Kit, OR
• RT2 SYBR Green Mastermix (2-Pack)
Call 1-888-503-3187 for more information
Email: support@SABiosciences.com
(2012 US and Canada only)
Webinar-related question?
QIAWEBINARS@QIAGEN.COM
- 74 -
Sample & Assay Technologies