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http://www.cedars-sinai.edu/Patients/Health-Conditions/Images/354031_Adv_Rheumatoid_Arthritis-2sm.jpg Inflammation Due To RA
Rheumatoid Arthritis Attack on flexible joints Bone Synovium Cartilage
Rheumatoid Arthritis Attack on flexible joints Bone Destruction Synovial Inflammation Bone Synovium Cartilage
Rheumatoid Arthritis Bone Destruction http://www.riversideonline.com/source/images/image_popup/mcdc7_arthritis_types.jpg Synovial Inflammation What causes it?
Osteoclast cells Bone destroyers
Surface Bound Protein Osteoclast Formation
osteoblast Osteoclast Formation
osteoblast Osteoclast Formation
Th17 Cells Type of T-helper Cells Major role in inflammation during RA Produce signaling molecules Present in the synovial fluid in RA patients
1,25 dimethydroxyvitamin D3 (1,25 (OH) 2 D3) Active Vitamin D Known inhibitor of bone resorption Vitamin D deficiency is common in RA patients Can inhibit osteoclast differentiation
Can osteoclast differentiation be induced in vitro by Th17 via cell to cell contact?
The Experiment [Osteoclast precursors + Th17 Cells] with and without [1,25 (OH) 2 D3] Measure osteoclast differentiation qPCR on mRNA of TRAP protein from osteoclasts
Since TRAP protein is produced only in osteoclasts, it can be used to measure osteoclast differentiation.
Quantitative real-time PCR Amplify DNA RNA primers target gene Fluorescence molecules “tag” DNA Detection Change in detection can be plotted and analyzed
DNA preparation mRNAcDNA RNA transcriptase
Priming DNA is heated, separating into single strands. RNA primers add to adjacent sequence on the 3’ end of target gene.
Extension DNA Polymerase extends new strand starting at the primers towards 5’ end.
Amplification Process is repeated until target gene is significantly detected.
Detection Sybr Green only attaches to double stranded DNA molecules. the tagged sequence will be detected.
Analysis Example GAPDH is used a reference gene Used in cell respiration Consistently expressed. GAPDH Target gene cDNA detected
Samples containing only osteoclast precursors Relative TRAP mRNA levels Modified from Kim et al
Reduced RANK expression Relative TRAP mRNA levels
In Rheumatoid Arthritis, Do Th17 Cells play a major role in osteoclast differentiation in vivo? Use T cell specific RANKL knockout mice. Collagen Induced Mouse Model
Inflammation Due To RA.
How do you identify and clone a gene of interest? Shotgun approach? Is there a better way?
Polymerase Chain Reaction PCR. PCR allows for amplification of a small piece of DNA. Some applications of PCR are in: –forensics (paternity testing, crimes)
RNA-Seq An alternative to microarray. Steps Grow cells or isolate tissue (brain, liver, muscle) Isolate total RNA Isolate mRNA from total RNA (poly.
Additional Powerful Molecular Techniques Synthesis of cDNA (complimentary DNA) Polymerase Chain Reaction (PCR) Microarray analysis Link to Gene Therapy.
PCR With PCR it is possible to amplify a single piece of DNA, or a very small number of pieces of DNA, over many cycles, generating millions of copies.
The polymerase chain reaction (PCR) rapidly
TACKLING OSTEOARTHRITIS -Research Tools At Our Disposal Mahita Kadmiel July 21, 2005.
DNA Replication DNA mRNA protein transcription translation replication Before each cell division the DNA must be replicated so each daughter cell can get.
Molecular Genetic Technologies Gel Electrophoresis PCR Restriction & ligation Enzymes Recombinant plasmids and transformation DNA microarrays DNA profiling.
Vectors Bacteria, viruses or liposomes into which DNA can be inserted. These can be used to grow genes, harvest the proteins they code for or deliver them.
Dr. Sumbul Fatma Department of Medical Biochemistry.
Kevin Chen. A method of amplifying or copying DNA fragments.
Analyzing your clone 1) FISH 2) “Restriction mapping” 3) Southern analysis : DNA 4) Northern analysis: RNA tells size tells which tissues or conditions.
-The methods section of the course covers chapters 21 and 22, not chapters 20 and 21 -Paper discussion on Tuesday - assignment due at the start of class.
RT-PCR analysis 생화학 실험 2 조교 : 이 선 민 내선 7699, 첨단과학기술관 201-B 호 신과학원 S438 호.
Gene Regulation: What it is, and how to detect it By Jordan, Jennifer, and Brian.
Polymerase chain reaction: Starting with VERY SMALL AMOUNTS OF DNA (sometimes a few molecules), one can amplify the DNA enough to detect it by electrophoresis.
Green with envy?? Jelly fish “GFP” Transformed vertebrates.
Polymerase Chain Reaction (PCR) Nahla Bakhamis. Multiple copies of specific DNA sequences; ‘Molecular Photocopying’
Restriction Nucleases Cut at specific recognition sequence Fragments with same cohesive ends can be joined.
PHYSICAL MAPPING AND POSITIONAL CLONING. Linkage mapping – Flanking markers identified – 1cM, for example Probably ~ 1 MB or more in humans Need very.
DNA-based Methods for Quantifying Microbes in Atmospheric Samples Tom Hill, Helen Ahern and Bruce Moffett University of East London.
Genomic DNA purification
AP Biology: Chapter 14 DNA Technologies
1. 2 VARIANTS OF PCR APPLICATIONS OF PCR MECHANICS OF PCR WHAT IS PCR? PRIMER DESIGN.
1 Chapter 2: DNA replication and applications DNA replication in the cell Polymerase chain reaction (PCR) Sequence analysis of DNA.
PCR, Gel Electrophoresis, and Southern Blotting
Research Techniques Made Simple: Polymerase Chain Reaction
Northern blotting & mRNA detection by qPCR - part 2.
Polymerase Chain Reaction (PCR)
V IRAL Q UEST Lesson 8: Reverse Transcription. Reverse Transcription Reverse Transcription converts RNA into complementary DNA. This DNA strand can.
Lecture ONE: Foundation Course Genetics Tools of Human Molecular Genetics I.
Lecturer: David. * Reverse transcription PCR * Used to detect RNA levels * RNA is converted to cDNA by reverse transcriptase * Then it is amplified.
Fig. S1 Beclin1, ATG3 and LC3B mRNA -real-time quantitative PCR HCT-116 HT-29.
Real-Time PCR mRNA quantification. What do mRNA levels tell us? DNA mRNA protein Reflect level of gene expression Information about cell response.
PCR quantitative en temps réel Lydie Pradel. PCR.
Methods in Cell Biology Cont. Sept. 24, Science Bomb 2 Unc-22: encodes a myofilament in C. elegans.
DNA Sequencing and Gene Analysis
The Polymerase Chain Reaction Some milestones In molecular biology recognised by the award of the Nobel prize.
Leukemia Cell Study Strode Note: Meaningless title.
Detecting DNA with DNA probes arrays. DNA sequences can be detected by DNA probes and arrays (= collection of microscopic DNA spots attached to a solid.
DNA (gene mutations, paternity, organs compatibility for transplantations) RNA Proteins (gene expression)
INTRODUCTION. INTRODUCTION Introduction In the past, amplifying (replication) of DNA was done in bacteria and took weeks. In 1971, paper in the.
Chapter 14 GENETIC TECHNOLOGY. A. Manipulation and Modification of DNA 1. Restriction Enzymes Recognize specific sequences of DNA (usually palindromes)
Transposon “Jumping Genes” Insertion Sequence
Polymerase Chain Reaction (PCR). PCR produces billions of copies of a specific piece of DNA from trace amounts of starting material. (i.e. blood, skin.
Polymerase Chain Reaction WORKSHOP (3)
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