DNA microarray is a technique that allows high-throughput analysis of gene expression. It involves depositing DNA fragments onto a glass slide and using fluorescent probes made from sample RNA to detect expression levels of thousands of genes simultaneously. The document discusses the basic principles and steps of DNA microarray, including sample preparation, hybridization, image analysis and data normalization. It also compares different microarray fabrication technologies and platforms, and discusses quality control considerations and limitations of the technique.
Next Generation Sequencing and its Applications in Medical Research - Frances...Sri Ambati
The so-called “next-generation” sequencing (NGS) technologies allows us, in a short time and in parallel, to sequence massive amounts of DNA, overcoming the limitations of the original Sanger sequencing methods used to sequence the first human genome. NGS technologies have had an enormous impact on biomedical research within a short time frame. This talk will give an overview of these applications with specific examples from Mendelian genomics and cancer research. #h2ony
Supporting Genomics in the Practice of Medicine by Heidi RehmKnome_Inc
View the webinar at http://www.knome.com/webinar-supporting-genomics-practice-medicine. In this presentation, Dr. Heidi Rehm, Chief Laboratory Director of the Laboratory for Molecular Medicine at Partners Healthcare and one of the Principal Investigators on ClinGen, elucidates the challenges of genomics in medicine and outlined the path to integrating large scale sequencing into clinical practice.
Avances en genética. Utilidad de la NGS y la bioinformática.BBK Innova Sarea
27 Octubre 2014. Presentación de Pablo Lapunzina, Director del Instituto de Medicina Genética Médica y Molecular (INGEMM), de IDIPAZ y de CEBERER, en la "Jornada Avances en Genética y Tecnología Social. La experiencia de la Fundación Síndrome de Dravet ".
Using methylation patterns to determine origin of biological material and ageQIAGEN
In this QIAGEN sponsored webinar, our guest speakers from the San Francisco Police Department (SFPD) Crime Lab and Florida International University (FIU) discuss their research on the potential of epigenetic methylation as a procedure for body fluid identification and age estimation from DNA left at crime scenes. Several approaches have been studied, including an analysis of methyl array data and an initial validation of procedures such as pyrosequencing and real-time PCR. The presentation focuses on a number of tissue-specific epigenetic markers for body fluid and age determination with a promise of future integration of these markers into the forensic lab due to the simplicity of analysis and the ease of application.
Learn more about the Pyrosequencing technology and our solutions at
https://www.qiagen.com/resources/technologies/pyrosequencing-resource-center/
The CRISPR/Cas9 system has emerged as one of the leading tools for modifying genomes of organisms ranging from E. coli to humans. Additionally, the simple gene targeting mechanism of CRISPR technology has been modified and adapted to other applications that include gene regulation, detection of intercellular trafficking, and pathogen detection. With a wealth of methods for introducing Cas9 and gRNAs into cells, it can be challenging to decide where to start. In this presentation, Dr Adam Clore describes the CRISPR mechanism and some of the most prominent uses for CRISPR, along with methods where IDT technologies can assist scientists in designing, testing, and executing a variety of CRISPR-mediated experiments. For more informaton, visit: http://www.idtdna.com/crispr
Next Generation Sequencing and its Applications in Medical Research - Frances...Sri Ambati
The so-called “next-generation” sequencing (NGS) technologies allows us, in a short time and in parallel, to sequence massive amounts of DNA, overcoming the limitations of the original Sanger sequencing methods used to sequence the first human genome. NGS technologies have had an enormous impact on biomedical research within a short time frame. This talk will give an overview of these applications with specific examples from Mendelian genomics and cancer research. #h2ony
Supporting Genomics in the Practice of Medicine by Heidi RehmKnome_Inc
View the webinar at http://www.knome.com/webinar-supporting-genomics-practice-medicine. In this presentation, Dr. Heidi Rehm, Chief Laboratory Director of the Laboratory for Molecular Medicine at Partners Healthcare and one of the Principal Investigators on ClinGen, elucidates the challenges of genomics in medicine and outlined the path to integrating large scale sequencing into clinical practice.
Avances en genética. Utilidad de la NGS y la bioinformática.BBK Innova Sarea
27 Octubre 2014. Presentación de Pablo Lapunzina, Director del Instituto de Medicina Genética Médica y Molecular (INGEMM), de IDIPAZ y de CEBERER, en la "Jornada Avances en Genética y Tecnología Social. La experiencia de la Fundación Síndrome de Dravet ".
Using methylation patterns to determine origin of biological material and ageQIAGEN
In this QIAGEN sponsored webinar, our guest speakers from the San Francisco Police Department (SFPD) Crime Lab and Florida International University (FIU) discuss their research on the potential of epigenetic methylation as a procedure for body fluid identification and age estimation from DNA left at crime scenes. Several approaches have been studied, including an analysis of methyl array data and an initial validation of procedures such as pyrosequencing and real-time PCR. The presentation focuses on a number of tissue-specific epigenetic markers for body fluid and age determination with a promise of future integration of these markers into the forensic lab due to the simplicity of analysis and the ease of application.
Learn more about the Pyrosequencing technology and our solutions at
https://www.qiagen.com/resources/technologies/pyrosequencing-resource-center/
The CRISPR/Cas9 system has emerged as one of the leading tools for modifying genomes of organisms ranging from E. coli to humans. Additionally, the simple gene targeting mechanism of CRISPR technology has been modified and adapted to other applications that include gene regulation, detection of intercellular trafficking, and pathogen detection. With a wealth of methods for introducing Cas9 and gRNAs into cells, it can be challenging to decide where to start. In this presentation, Dr Adam Clore describes the CRISPR mechanism and some of the most prominent uses for CRISPR, along with methods where IDT technologies can assist scientists in designing, testing, and executing a variety of CRISPR-mediated experiments. For more informaton, visit: http://www.idtdna.com/crispr
Making genome edits in mammalian cellsChris Thorne
Looking at the kind of modifications that can be made in mammalian cells, and how at Horizon moving to a haploid model system has significantly improved efficiency of both editing and validation
This year's 3rd Annual TCGC: The Clinical Genome Conference, held June 10-12, 2014 in San Francisco, is a three-day event that weaves together the science of sequencing and the business of implementing genomics in the clinic. It uniquely illustrates the mutual influence of those areas and the need to therefore consider the needs, challenges and opportunities of both - from next-generation sequencing and variant interpretation to insurance reimbursement and electronic health records - throughout the entire research process.Learn more at http://www.clinicalgenomeconference.com
"SNP and STR analysis using NGS
Niels Morling, MD DMSc
Professor of Forensic Genetics
Chairman & Director
Department of Forensic Medicine
Faculty of Health and Medical Sciences
University of Copenhagen
Denmark"
Developing a Rapid Clinical Sequencing System to Classify Meningioma: Meet th...QIAGEN
Meningioma’s display a broad spectrum of clinical, histological and cytogenetic features even within the same WHO grade often posing a challenge for classification and prognostic stratification. In this webinar, we will describe our experience of using targeted amplicon sequencing to develop rapid clinical sequencing system to identify and confirm the meningioma genotype in just two weeks. In addition the details of the three meningioma categories and the genes involved will be discussed.
Clinical molecular diagnostics for drug guidanceNikesh Shah
1. Be familiar with next generation molecular diagnostic techniques that can provide guidance in clinical decision making
2. Identify the utility of these diagnostic approaches with some examples
3. Be aware of the challenges that exist in implementing these tools as part of the routine clinical decision making process, especially in resource limited settings
GTC group 8 - Next Generation SequencingYanqi Chan
DNA sequencing is the process of determining the precise order of nucleotides within a DNA molecule. Discuss the application of next generation sequencing in cancer treatment.
understanding of the human immune system, and thereby cancer immunology.
αβT-cells are the primary constituents of human cell-mediated adaptive immunity.
The antigen specificity of each αβT-cell is encoded in the 500-600 bp transcript
encompassing the variable portion of the rearranged TCRα and TCRβ subunits,
which can be read via NGS in a process termed repertoire sequencing. Until now,
the main challenge the field faces is the lack of a technology that can provide a
contiguous read of 600 bp to minimize the complexity of designing bias-prone
primers and informatics challenges of stitching short reads. Here we leverage the
long read capability of Ion 530™ chip to comprehensively sequence all three CDR
domains of the TCRβ chain. The Ion 530™ chip offers greater than 15 M productive
reads, allowing a multiplex of 2-4 samples with sufficient coverage for most repertoire
profiling studies. Initial testing with leukocyte total RNA demonstrates that this
multiplex PCR assay produced repertoires that were much more similar to data
derived from 5’-RACE protocol than the commonly used BIOMED-2 primer set. This
result suggested that the use of long reads minimizes bias by allowing targeting of
less variable regions. To further assess the performance of the assay, we designed a
model system of 30 plasmid controls containing common human T-cell CDR3
sequences. Each plasmid was amplified individually and sequenced to confirm the
detection of a single clonal population. Analytical sensitivity of the assay and
accuracy of the accompanied analysis solution were further evaluated by spiking in
plasmid concentrations from 10 pg to 0.0001 pg (5 million to 50 copies) in a
background of 100 ng cDNA reverse transcribed from leukocyte total RNA. Results
showed the assay offers linearity over 5 orders of magnitude of decreasing input
concentration. In summary, we have demonstrated a NGS workflow for TCRβ
sequencing that offers multiplex flexibility on Ion S5 with sample to answer in less
than 48 hours.
Visualizing Human Stem Cell Dynamics by Multicolor, Multiday High-Content Mic...InsideScientific
Visualizing the complex spatiotemporal dynamics of human stem cells as they proliferate and make cell fate decisions is key to improve our understanding of how to robustly engineer differentiated tissues for therapeutic applications.
In this webinar, Dr. Rafael Carazo Salas describes multicolor, multiday high-content microscopy pipelines that his group has recently developed to visualize the dynamical cell fate changes of human Pluripotent Stem Cells (hPSCs). In particular, he reviews the integrated experimental and computational approaches that his group has established, including novel “live” reporters of cell fate and multi-reporter hPSC lines generated by CRISPR/Cas9 allowing multiplexed monitoring of cell proliferation and fate dynamics, and exemplify the biological discoveries they are enabling.
Key Topics Include:
- Visualizing how human Pluripotent Stem Cells (hPSCs) proliferate and undergo early differentiation in vitro, by high content microscopy
- Learning about experimental and computational pipelines that enable cell fate monitoring at the collective and single-cell level
- Learning about novel “live” reporters of hPSC cell fate
Plant Chromosomes: European Cytogeneticists outline: Trude Schwarzacher and P...Pat (JS) Heslop-Harrison
An overview of plant molecular cytogenetics. The lecture Trude Schwarzacher presented to the ECA conference Strasbourg in July 2015 is http://www.slideshare.net/PatHeslopHarrison/trude-schwarzacher
Transgene-free CRISPR/Cas9 genome-editing methods in plantsCIAT
"Transgene-free CRISPR/Cas9 genome-editing methods in plants" by Matthew R. Willmann, Ph.D. Director, Plant Transformation Facility College of Agriculture and Life Sciences, School of Integrative Plant Science, Cornell University.
COVID-19: Biology, Transmission, and DetectionAlejandroAlRuiz
A short PowerPoint for those interested in learning more about the biology of SARS-CoV-2, how it is transmitted, and what are the current methods of detection. This PowerPoint also demonstrates how we can begin to teach about SARS-CoV-2 in our classrooms.
Next generation Sequencing or massive parallel sequencing is a high throughput approach to sequence genetic material using the concept of massively parallel processing. It is also called second generation sequencing.This enables researchers a wide variety of applications & study biological systems.
Making genome edits in mammalian cellsChris Thorne
Looking at the kind of modifications that can be made in mammalian cells, and how at Horizon moving to a haploid model system has significantly improved efficiency of both editing and validation
This year's 3rd Annual TCGC: The Clinical Genome Conference, held June 10-12, 2014 in San Francisco, is a three-day event that weaves together the science of sequencing and the business of implementing genomics in the clinic. It uniquely illustrates the mutual influence of those areas and the need to therefore consider the needs, challenges and opportunities of both - from next-generation sequencing and variant interpretation to insurance reimbursement and electronic health records - throughout the entire research process.Learn more at http://www.clinicalgenomeconference.com
"SNP and STR analysis using NGS
Niels Morling, MD DMSc
Professor of Forensic Genetics
Chairman & Director
Department of Forensic Medicine
Faculty of Health and Medical Sciences
University of Copenhagen
Denmark"
Developing a Rapid Clinical Sequencing System to Classify Meningioma: Meet th...QIAGEN
Meningioma’s display a broad spectrum of clinical, histological and cytogenetic features even within the same WHO grade often posing a challenge for classification and prognostic stratification. In this webinar, we will describe our experience of using targeted amplicon sequencing to develop rapid clinical sequencing system to identify and confirm the meningioma genotype in just two weeks. In addition the details of the three meningioma categories and the genes involved will be discussed.
Clinical molecular diagnostics for drug guidanceNikesh Shah
1. Be familiar with next generation molecular diagnostic techniques that can provide guidance in clinical decision making
2. Identify the utility of these diagnostic approaches with some examples
3. Be aware of the challenges that exist in implementing these tools as part of the routine clinical decision making process, especially in resource limited settings
GTC group 8 - Next Generation SequencingYanqi Chan
DNA sequencing is the process of determining the precise order of nucleotides within a DNA molecule. Discuss the application of next generation sequencing in cancer treatment.
understanding of the human immune system, and thereby cancer immunology.
αβT-cells are the primary constituents of human cell-mediated adaptive immunity.
The antigen specificity of each αβT-cell is encoded in the 500-600 bp transcript
encompassing the variable portion of the rearranged TCRα and TCRβ subunits,
which can be read via NGS in a process termed repertoire sequencing. Until now,
the main challenge the field faces is the lack of a technology that can provide a
contiguous read of 600 bp to minimize the complexity of designing bias-prone
primers and informatics challenges of stitching short reads. Here we leverage the
long read capability of Ion 530™ chip to comprehensively sequence all three CDR
domains of the TCRβ chain. The Ion 530™ chip offers greater than 15 M productive
reads, allowing a multiplex of 2-4 samples with sufficient coverage for most repertoire
profiling studies. Initial testing with leukocyte total RNA demonstrates that this
multiplex PCR assay produced repertoires that were much more similar to data
derived from 5’-RACE protocol than the commonly used BIOMED-2 primer set. This
result suggested that the use of long reads minimizes bias by allowing targeting of
less variable regions. To further assess the performance of the assay, we designed a
model system of 30 plasmid controls containing common human T-cell CDR3
sequences. Each plasmid was amplified individually and sequenced to confirm the
detection of a single clonal population. Analytical sensitivity of the assay and
accuracy of the accompanied analysis solution were further evaluated by spiking in
plasmid concentrations from 10 pg to 0.0001 pg (5 million to 50 copies) in a
background of 100 ng cDNA reverse transcribed from leukocyte total RNA. Results
showed the assay offers linearity over 5 orders of magnitude of decreasing input
concentration. In summary, we have demonstrated a NGS workflow for TCRβ
sequencing that offers multiplex flexibility on Ion S5 with sample to answer in less
than 48 hours.
Visualizing Human Stem Cell Dynamics by Multicolor, Multiday High-Content Mic...InsideScientific
Visualizing the complex spatiotemporal dynamics of human stem cells as they proliferate and make cell fate decisions is key to improve our understanding of how to robustly engineer differentiated tissues for therapeutic applications.
In this webinar, Dr. Rafael Carazo Salas describes multicolor, multiday high-content microscopy pipelines that his group has recently developed to visualize the dynamical cell fate changes of human Pluripotent Stem Cells (hPSCs). In particular, he reviews the integrated experimental and computational approaches that his group has established, including novel “live” reporters of cell fate and multi-reporter hPSC lines generated by CRISPR/Cas9 allowing multiplexed monitoring of cell proliferation and fate dynamics, and exemplify the biological discoveries they are enabling.
Key Topics Include:
- Visualizing how human Pluripotent Stem Cells (hPSCs) proliferate and undergo early differentiation in vitro, by high content microscopy
- Learning about experimental and computational pipelines that enable cell fate monitoring at the collective and single-cell level
- Learning about novel “live” reporters of hPSC cell fate
Plant Chromosomes: European Cytogeneticists outline: Trude Schwarzacher and P...Pat (JS) Heslop-Harrison
An overview of plant molecular cytogenetics. The lecture Trude Schwarzacher presented to the ECA conference Strasbourg in July 2015 is http://www.slideshare.net/PatHeslopHarrison/trude-schwarzacher
Transgene-free CRISPR/Cas9 genome-editing methods in plantsCIAT
"Transgene-free CRISPR/Cas9 genome-editing methods in plants" by Matthew R. Willmann, Ph.D. Director, Plant Transformation Facility College of Agriculture and Life Sciences, School of Integrative Plant Science, Cornell University.
COVID-19: Biology, Transmission, and DetectionAlejandroAlRuiz
A short PowerPoint for those interested in learning more about the biology of SARS-CoV-2, how it is transmitted, and what are the current methods of detection. This PowerPoint also demonstrates how we can begin to teach about SARS-CoV-2 in our classrooms.
Next generation Sequencing or massive parallel sequencing is a high throughput approach to sequence genetic material using the concept of massively parallel processing. It is also called second generation sequencing.This enables researchers a wide variety of applications & study biological systems.
Towards Precision Medicine: Tute Genomics, a cloud-based application for anal...Reid Robison
Tute Genomics is cloud-based software that can rapidly analyze entire human genomes. The cost of whole genome sequencing is dropping rapidly and we are in the middle of a genomic revolution. Tute is opening a new door for personalized medicine by helping researchers & healthcare organizations analyze human genomes.
A DNA microarray (also commonly known as DNA chip or biochip) is a collection of microscopic DNA spots attached to a solid surface.
The core principle behind microarrays is hybridization between two DNA strands, the property of complementary nucleic acid sequences to specifically pair with each other by forming hydrogen bonds between complementary nucleotide base pairs.
Advances and Applications Enabled by Single Cell TechnologyQIAGEN
Over the past 5 years, single-cell genomics have become a powerful technology for studying small samples and rare cells, and for dissecting complex populations such as heterogeneous tumors. Single-cell technology is enabling many new insights into diverse research areas from oncology, immunology and microbiology to neuroscience, stem cell and developmental biology. This webinar introduces single-cell technology and summarizes the newest scientific applications in various research areas, all in the context of current literature.
micro teaching on communication m.sc nursing.pdfAnurag Sharma
Microteaching is a unique model of practice teaching. It is a viable instrument for the. desired change in the teaching behavior or the behavior potential which, in specified types of real. classroom situations, tends to facilitate the achievement of specified types of objectives.
Tom Selleck Health: A Comprehensive Look at the Iconic Actor’s Wellness Journeygreendigital
Tom Selleck, an enduring figure in Hollywood. has captivated audiences for decades with his rugged charm, iconic moustache. and memorable roles in television and film. From his breakout role as Thomas Magnum in Magnum P.I. to his current portrayal of Frank Reagan in Blue Bloods. Selleck's career has spanned over 50 years. But beyond his professional achievements. fans have often been curious about Tom Selleck Health. especially as he has aged in the public eye.
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Introduction
Many have been interested in Tom Selleck health. not only because of his enduring presence on screen but also because of the challenges. and lifestyle choices he has faced and made over the years. This article delves into the various aspects of Tom Selleck health. exploring his fitness regimen, diet, mental health. and the challenges he has encountered as he ages. We'll look at how he maintains his well-being. the health issues he has faced, and his approach to ageing .
Early Life and Career
Childhood and Athletic Beginnings
Tom Selleck was born on January 29, 1945, in Detroit, Michigan, and grew up in Sherman Oaks, California. From an early age, he was involved in sports, particularly basketball. which played a significant role in his physical development. His athletic pursuits continued into college. where he attended the University of Southern California (USC) on a basketball scholarship. This early involvement in sports laid a strong foundation for his physical health and disciplined lifestyle.
Transition to Acting
Selleck's transition from an athlete to an actor came with its physical demands. His first significant role in "Magnum P.I." required him to perform various stunts and maintain a fit appearance. This role, which he played from 1980 to 1988. necessitated a rigorous fitness routine to meet the show's demands. setting the stage for his long-term commitment to health and wellness.
Fitness Regimen
Workout Routine
Tom Selleck health and fitness regimen has evolved. adapting to his changing roles and age. During his "Magnum, P.I." days. Selleck's workouts were intense and focused on building and maintaining muscle mass. His routine included weightlifting, cardiovascular exercises. and specific training for the stunts he performed on the show.
Selleck adjusted his fitness routine as he aged to suit his body's needs. Today, his workouts focus on maintaining flexibility, strength, and cardiovascular health. He incorporates low-impact exercises such as swimming, walking, and light weightlifting. This balanced approach helps him stay fit without putting undue strain on his joints and muscles.
Importance of Flexibility and Mobility
In recent years, Selleck has emphasized the importance of flexibility and mobility in his fitness regimen. Understanding the natural decline in muscle mass and joint flexibility with age. he includes stretching and yoga in his routine. These practices help prevent injuries, improve posture, and maintain mobilit
NVBDCP.pptx Nation vector borne disease control programSapna Thakur
NVBDCP was launched in 2003-2004 . Vector-Borne Disease: Disease that results from an infection transmitted to humans and other animals by blood-feeding arthropods, such as mosquitoes, ticks, and fleas. Examples of vector-borne diseases include Dengue fever, West Nile Virus, Lyme disease, and malaria.
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Rasamanikya is a excellent preparation in the field of Rasashastra, it is used in various Kushtha Roga, Shwasa, Vicharchika, Bhagandara, Vatarakta, and Phiranga Roga. In this article Preparation& Comparative analytical profile for both Formulationon i.e Rasamanikya prepared by Kushmanda swarasa & Churnodhaka Shodita Haratala. The study aims to provide insights into the comparative efficacy and analytical aspects of these formulations for enhanced therapeutic outcomes.
Adv. biopharm. APPLICATION OF PHARMACOKINETICS : TARGETED DRUG DELIVERY SYSTEMSAkankshaAshtankar
MIP 201T & MPH 202T
ADVANCED BIOPHARMACEUTICS & PHARMACOKINETICS : UNIT 5
APPLICATION OF PHARMACOKINETICS : TARGETED DRUG DELIVERY SYSTEMS By - AKANKSHA ASHTANKAR
Local Advanced Lung Cancer: Artificial Intelligence, Synergetics, Complex Sys...Oleg Kshivets
Overall life span (LS) was 1671.7±1721.6 days and cumulative 5YS reached 62.4%, 10 years – 50.4%, 20 years – 44.6%. 94 LCP lived more than 5 years without cancer (LS=2958.6±1723.6 days), 22 – more than 10 years (LS=5571±1841.8 days). 67 LCP died because of LC (LS=471.9±344 days). AT significantly improved 5YS (68% vs. 53.7%) (P=0.028 by log-rank test). Cox modeling displayed that 5YS of LCP significantly depended on: N0-N12, T3-4, blood cell circuit, cell ratio factors (ratio between cancer cells-CC and blood cells subpopulations), LC cell dynamics, recalcification time, heparin tolerance, prothrombin index, protein, AT, procedure type (P=0.000-0.031). Neural networks, genetic algorithm selection and bootstrap simulation revealed relationships between 5YS and N0-12 (rank=1), thrombocytes/CC (rank=2), segmented neutrophils/CC (3), eosinophils/CC (4), erythrocytes/CC (5), healthy cells/CC (6), lymphocytes/CC (7), stick neutrophils/CC (8), leucocytes/CC (9), monocytes/CC (10). Correct prediction of 5YS was 100% by neural networks computing (error=0.000; area under ROC curve=1.0).
Title: Sense of Smell
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the primary categories of smells and the concept of odor blindness.
Explain the structure and location of the olfactory membrane and mucosa, including the types and roles of cells involved in olfaction.
Describe the pathway and mechanisms of olfactory signal transmission from the olfactory receptors to the brain.
Illustrate the biochemical cascade triggered by odorant binding to olfactory receptors, including the role of G-proteins and second messengers in generating an action potential.
Identify different types of olfactory disorders such as anosmia, hyposmia, hyperosmia, and dysosmia, including their potential causes.
Key Topics:
Olfactory Genes:
3% of the human genome accounts for olfactory genes.
400 genes for odorant receptors.
Olfactory Membrane:
Located in the superior part of the nasal cavity.
Medially: Folds downward along the superior septum.
Laterally: Folds over the superior turbinate and upper surface of the middle turbinate.
Total surface area: 5-10 square centimeters.
Olfactory Mucosa:
Olfactory Cells: Bipolar nerve cells derived from the CNS (100 million), with 4-25 olfactory cilia per cell.
Sustentacular Cells: Produce mucus and maintain ionic and molecular environment.
Basal Cells: Replace worn-out olfactory cells with an average lifespan of 1-2 months.
Bowman’s Gland: Secretes mucus.
Stimulation of Olfactory Cells:
Odorant dissolves in mucus and attaches to receptors on olfactory cilia.
Involves a cascade effect through G-proteins and second messengers, leading to depolarization and action potential generation in the olfactory nerve.
Quality of a Good Odorant:
Small (3-20 Carbon atoms), volatile, water-soluble, and lipid-soluble.
Facilitated by odorant-binding proteins in mucus.
Membrane Potential and Action Potential:
Resting membrane potential: -55mV.
Action potential frequency in the olfactory nerve increases with odorant strength.
Adaptation Towards the Sense of Smell:
Rapid adaptation within the first second, with further slow adaptation.
Psychological adaptation greater than receptor adaptation, involving feedback inhibition from the central nervous system.
Primary Sensations of Smell:
Camphoraceous, Musky, Floral, Pepperminty, Ethereal, Pungent, Putrid.
Odor Detection Threshold:
Examples: Hydrogen sulfide (0.0005 ppm), Methyl-mercaptan (0.002 ppm).
Some toxic substances are odorless at lethal concentrations.
Characteristics of Smell:
Odor blindness for single substances due to lack of appropriate receptor protein.
Behavioral and emotional influences of smell.
Transmission of Olfactory Signals:
From olfactory cells to glomeruli in the olfactory bulb, involving lateral inhibition.
Primitive, less old, and new olfactory systems with different path
Integrating Ayurveda into Parkinson’s Management: A Holistic ApproachAyurveda ForAll
Explore the benefits of combining Ayurveda with conventional Parkinson's treatments. Learn how a holistic approach can manage symptoms, enhance well-being, and balance body energies. Discover the steps to safely integrate Ayurvedic practices into your Parkinson’s care plan, including expert guidance on diet, herbal remedies, and lifestyle modifications.
3. Approaches for Characterizing Differential Gene
Expression
Low-throughput or Single Gene Methods
High-throughput or Large-Scale Methods
4. The Hybridization of Complementary
Strands of DNA/RNA
Is the Underlying Principle of All
Methods of Differential Gene Expression.
5. Single Gene Methods
Northern Blotting, cumbersome, time-consuming
Nuclease protection, at least 10 fold more sensitive
Quantitative RT-PCR, state of the art
6. High-throughput Methods
Serial Analysis of Gene Expression (SAGE)
Rapid Analysis of Gene Expression (RAGE)
Representational Difference Analysis (RDA)
Suppression Subtractive Hybridization (SSH)
Differential screening (plus/minus screening)
Differential Display (DD)
DNA Microarray
Comprehensive evaluation
400,000 Northern Blotting
7. What is DNA Microarray?
A large number of genes deposited onto a glass slide (large scale dot blot)
The RNA sample is RT with simultaneous incorporation of label,
resulting in labeled cDNA.
Microarray slides serve as hybridization targets for labeled cDNA
Reverse Northern blotting
Patrick O Brown
Mark Schena
8. Basic Steps in Performing a DNA Microarray Experiments
1- Processing cDNA clones to generate print-ready material
2-Printing cDNA clones (or oligonucleotide) onto a substrate
3-Sample RNA isolation
4-Preparation of the probe (e.g. cDNA synthesis and labeling, RT reaction)
5- Hybridization of labeled probe DNA to the DNA arrayed on the substrate
6-Image acquisition, image analysis and data analysis
9.
10. Microarray Fabrication Technologies
In Situ Synthesis of Nucleic Acid (Chip ,GeneChip,oligonucleotide array)
15-20 different 25-mer oligonucleotides
Exogenous Deposition of cDNA (cDNA, spotted array)
Single DNA fragments, greater 0.5 Kb
11.
12. Common Approaches for Microarray Fabrication
2-Contact printing (Patrick O Brown,Stanford University)
3- Non-Contact Printing (Pin and Ring, Bubble Jet, Ink Jet)
1- Photolithography (Affymetrix, Oligonucleotide Microarray)
13. What to spot?
As many known genes as possible
Genes that are most relevant
A combination of both approaches
Publicity available clones (IMAGE)
In_house derived (SSH)
Custom made/purchased libraries
14. Analysis of Gene Expression
Monitoring Changes in Genomic
DNA
Gene Discovery, Sequencing and Pathway Analysis
When to use Microarray
15. Analysis of Gene Expression
1- Different tissues or different developmental states
2- Normal or diseased states
3- Exposure to drugs or different physiological conditions
16. Monitoring Changes in Genomic DNA
Hybridization to oligonucleotide is sensitive in
detection of single-nucleotide mismatches
Single Nucleotide Polymorphisms (SNPs)
High Density Oligonucleotide Array
Cancer cells typically exhibit genomic instability
17. Detailed Protocols
Stanford University
Albert Einstein College of Medicine
NHGRI
Cold Spring Harbor Laboratory
Collection of Protocols
TIGR Protocols
www.cmgm.stanford.edu/pbrown/
www.sequence.aecom.yu.edu/bioinf/microarray/protocol.html
www.nhgri.gov/DIR/LCG/15K/HTML/protocol.html
www.nucleus.cshl.org/wigler
www.protocol-online.net/molbio/DNA/dna_microarray.html
www.tigr.org/tdb/microarray
18. Two basic substrates commonly used for cDNA printing
are glass and membrane filters
Chemically treated microscope glass slides are the most
widely used support
Microarray, Microscope Slide,80000 Spots, 10000-20000 Spots
Macroarray, Nylon Membrane, 500,-18000 Spots
Micro or Macro
19. RNA Preparation
No difference between total RNA or mRNA
Type of tissue might have profound effect on extraction
process. 100 -200 µg of RNA is needed/slide
Laser captured microdissection (LCM) , incorporation of a
PCR step
20. Sample Labeling
Most microarray utilize two fluorophores,
Cyanine3(Green emission) and Cyanine5 (Red emission)
They have different size and different ability
for incorporation in cDNA
A single round of transcription is used to generate
a labeled cDNA probe (RT-PCR)
21. Data Analysis
Normalization
First step is during scanning, when sensitivity of
detection is adjusted by the laser voltage
Gene expression value can be expressed relative
to the expression of housekeeping genes
In the absence of control genes, normalization to the median
microarray value is popular
No consensus, ANOVA
Clustering (categorizing genes according to their pattern of expression)
22. Analyzed gene changes are often expressed as a fold increase
either greater than twofold or less than 0.5 fold (DeRisi)
How Much is Significant???
With a large number of microarrays, small changes can be statistically valid
Elcock et al. detected 1.1 fold changes with 95 % confidence interval when
each experimental sample was hybridized to
seven microarray slides (with two replicate spots for each gene)
Derisi et al.Nat Genet 1996:14:457-60
23. Housekeeping genes
These are genes that are expressed constitutively and their level of
expression is thought to be stable, regardless of the sample used (β
Actin, Cyclophilin, GAPDH)
DeRisi used 90 housekeeping genes and found that changes that
were <0.5 and > 2.4 were acceptable
β Actin is one of the most commonly used housekeeping genes
and it has been shown to be downregulated in heat shock experiments
In fact, there is an appreciable amount of literature available to
suggest that there is no such thing as housekeeping gene
24. DNA microarray represents a developing technology, there remain
substantial obstacles in the design and analysis of these microarray
There are no globally accepted rules or standards
for performing controlled microarray experiments
A good experiments include more control component then
the real comparison
Accuracy and Precision
25. Principles of Q.C in DNA Microarray
Down-Scaling of an experiment makes it generally
sensitive to external and internal fluctuation
Replication of each experiments on multiple array
Dual labeling, swapping the dyes for control and treated
sample
Using a large number of controls on every array
26. Controls
mRNA from genes that are not homologous to the organism understudy (Arabidopsis)
cDNA from the organism with high, medium and
low expression represented on the array (sensitivity)
Cold DNA (e.g., calf thymus DNA, yeast tRNA)
is added to block nonspecific annealing
Spots of DNA from another organism whose
mRNA is not represented in the sample (Background)
Total genomic DNA or cDNA clones of common contaminant such
as E.Coli and yeast are represented in the array to monitor for contamination
27. Ontario Cancer Institute
Spotted Array
Advantages
Gene discovery
Optimal size(specific hybridization)
Available technology
Disadvantages
Clones processing is cumbersome
Lower density than chips
Cross hybridization(repetitive sequence)
28. Affymetrix Genechip
Biotinylated cRNA is synthesized from cDNA
phycoerthrin linked to avidin is used for labeling
Each sample hybridized separately
Advantages
High density chip
Consistent and uniform geometry
Single Nucleotide Polymorphisms
No need for maintaining cDNA clones
Disadvantages
Sequence data required
Oligonucleotid selection rules
are not well defined
Not best target for hybridization
Expensive
29. Rajeevan et al. estimated that 30% of
microarray results are false-positive
Rajeevan et al. J Mol Diag 2001-3-26-31
Microarray findings should be confirmed,at least
by one of the low-throughput gene expression methods
DNA microarray technology is in its infancy
Application of microarray in diabetes
is not born or at most is premature
30. Many genes are expressed constitutively and regulation
of their function is at the translational or posttranslational
(ApoB ,CFTR, TCR)
To date, there has been a relatively poor correlation
between gene and protein expression.
It is likely that global proteome analysis provides a better
representation of the phenotype than does gene expression analysis
32. Mouse Genechip or spotted microarray
Systematic evaluation of insulin signaling and dyslipidemia
different tissue,time course
TZD or other drugs
Parallel study with protoemic
What can we do?
33. Expressed Sequence Tag (EST)
A Partial DNA squence derived from a cDNA clone
enough to identify the transcript which the cDNA was derived
55% of cardiovascular ESTs matches to known genes
25% with other ESTs and 20% remain unmatch(novel)
2 million human ESTs deposited in GeneBank
and used as substrate for DAN microarray
34. C.C Liew,(1994) sequenced 3500 ESTs representing 3100
cDNA from adult human heart(First cadiovascular catalogue of genes)
The number of cardiovascular ESTs increased to 85,000 (1997)
The latest number(2001) is 111,224 cardiovascular ESTs
C.C.Liew:PNAS,1994;91-10645-10649
C.C.Liew: Circulation, 1997;96:4146-4203
C.C.Liew: J Mol Cell Cardiol, 2001,33,1879-1886
The largest cardiovascular cDNA microarray constructed (10,368 ESTs)
C.C.Liew. BBRC, 2000,280-964-969
Cadiovascular ESTs
35. The number of genes encoded by the Human genome has been
estimated ∼ 32,000 - 38,000.
Between 21,000 - 27,000 genes are expressed in the cardiovascular system
Lack of information
No cDNA Library for Atherosclerotic plaques
Only 5% of total ESTs deposited in GeneBank derived from cardiovscular tissue
ESTs from cardiovascular tissues or cell type
or from diseased specimens remain limited
36. Cardiovascular EST data from most model organisms are almost nonexistent
The construction of cardiovascular gene databases at different
stages of pathalogy cast light on the complex genetic
mechanisms underlying disease of cadiovascular system
DNA microarray technology is in infancy
DNA microarray in atherosclerosis was not
born or at least is premature
Premature
37. First study dealing with differential gene expression in whole-mount
specimens of rupture plaques using macroarray
Suppression Subtractive Hybridization (SSH) technique isolates low abundant
sequence that might not be isolated by use of microarray technology
Mammalian mRNA population
20% Abundant transcript (1000-12000 copies/cell)
25% Medium abundant (100-1000 copies/cell)
% 50 small number copies (< 13 copies/cell)
Mammalian mRNA encoding proteins that regular cellular
behavior are expressed at low abundence
39. Prelipin is unlikely to be the sole marker of rupture
The author used only 10% of differentially expressed gene for doing macroarray
A large effort at macroarray and then sequencing would have yield more differences
An alternative would be to hybridized the subtractand against a large array
Other alternative is the isolation of cell type-specific genes
(LCM) rather than plaque-type-specific genes
40. Perilipin was the known gene that unregulated (confirmed by RT-PCR) 8 of 10
ruptured plaques expressed prelipin while expression was absent in 10 stable plaque
Prelipin is a protein which present on the surface layer of
intracellular lipid droplets in adipocyte and prevent lipolysis
They speculated that this will result in increased lipid
retention and plaque destabilization
β actin was down regulated in ruptured plaques
The down regulation of one gene was not confirmed by RT-PCR
41. K.j.Haley et al. Treated cultured Human aortic SMC with TNFα and
used DNA microarray with 8600 genes to monitor gene expression
Marked increase in eotaxin confirmed with northern blotting
Immunohistochemical analysis demonstrated overexpression of
eotaxin and its receptor in the Human atheroma(SMC)
Circulation;2000:102:2185-2189 Bostom-Harvard
42. McCaffrey et al. compared transcript profile of fibrous cap vs adjacent media
of 13 patients ,using macroarray (membrane 588 known genes)
Early growth response gene(Egr-1) was highly
expressed in lesion (confirmed by RT-PCR)
Many Erg-1 inducible genes including PDGF , TGF-β and ICAM-1
were also strongly elevated in the lesion
Immunocytochemistry indicated that Egr-1 was expressed in SMC
β ACTIN and GAPDH were use as houskeeping gene
J.C.I 2000,105:653-662 Cornell University
43. L.D Adams, S.M Schwartz, University of Washington
Adams et al. Compared gene expression of media of aorta and
vena cava, using cDNA microarray of 4048 known genes
68 genes had consistent elevation in message expression the aorta
The most differentially gene was Regulator of G P rotein Signaling (RGS5)
Northern analysis and in situ hybridization were used to confirm the results
Circulation Research 2000.8.623
Role of Lipid Rafts in AMPA Receptor Trafficking and Synaptic Plasticity
Last ten years has been witness of emerging the concept of lipid rafts which has
changed and revolutionized the classical two-dimensional "fluid mosaic" model of
plasma membrane (Singer & Nicolson 1972). The new plasma membrane model or so
called "liquid-ordered" membrane is based on the existence of organized, detergent
resistance discrete detergent resistance microdomain of plasma membrane named lipid
rafts. Rafts are membrane subdomains, enriched in cholesterol and sphingolipids. These
microdomains act as plat forms for conducting a variety of cellular functions, such as
vesicular trafficking and signal transduction (recently reviewed by Simons K, Toomre D,
Nature Reviews, 2000, 1:31-39; Galbiati F., et al. Cell, 2001, 106:403-411).
Recent data supports that manipulation of cellular lipid composition especially
cholesterol and fatty acid contents of plasma membrane bilayer disrupt lipid
microdomains integrity, which can subsequently modulate signal transduction and
membrane trafficking. There are several classical methods to disrupt rafts integrity
including cholesterol sequestration (by using antibiotics such as filipin or nistatin; or by
using pore-forming agents such as saponin or digitonin), cholesterol depletion (by
methyl-β -cyclodextrin), inhibition of cholesterol synthesis (by statins), and perturbation
of raft stability (by using exogenous cholesterol, exogenous gangliosides, exogenous
polyunsaturated fatty acids).
Several important enzymes and signaling proteins such as insulin receptors,
PDGF, eNOS, CD36, src-family of tyrosine kinases are localized in lipid rafts (Ref: ).
More recently Suzuki et al. (Suzuki T., et al. 2001, Mol. Brain. Res. 89:20-28) reported
evidences for localization of AMPA-type glutamate receptors in the dendritic rafts.
Glutamate receptors (AMPAs, NMDARs) activities are essential to many neurological
functions. Overactivity of these receptors can cause neurological death as a result of
excitotoxicity. Excitotoxicity is a key event leading to neuronal injury in stroke patients.
Recent evidence supports central role of AMPA receptors in the pathologies caused by
brain ischemia. Although the underlying mechanism (s) are not fully understood,
modulating AMPA receptors have been shown to be neuroprotoctive. Therefore
44. R.M Lawn et al. examined the response of macrophages to exposure to
oxidized LDL, using microarray containing 10000 Human genes
268 genes were found to be at least twofold regulated
Real Time RT-PCR was used to confirm the results
Orphan nuclear receptors (PPARγ, LXR and RXR) and ABC1 were
among genes which unregulated after exposure
J.B.C 2000:275;48, 37324-37332
45. L.A Mcintire et al. identified 52 genes with altered expression under shear stress
Using DNA microarray in primary human umbilical vein endothelial cells
Significant increases in mRNA levels for 32 and significant
decreases in expression for 20 genes were reported
The most enhanced genes were cytocromes P45 1A1 and 1B1
and human prostaglandin transporter
Most dramatically decreased were connective tissue growth factor and endotheline-1
Northern blot analysis confirmed the results obtained on microarray
PNAS2001, 98:8955-8960 Rice University