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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
Research Techniques Made Simple: Polymerase Chain Reaction
Polymerase Chain Reaction (PCR)
PCR, Gel Electrophoresis, and Southern Blotting
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.
DNA Technology & Gene Mapping Biotechnology has led to many advances in science and medicine including the creation of DNA clones via recombinant clones,
TACKLING OSTEOARTHRITIS -Research Tools At Our Disposal Mahita Kadmiel July 21, 2005.
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.
Real-Time PCR mRNA quantification. What do mRNA levels tell us? DNA mRNA protein Reflect level of gene expression Information about cell response.
DNA Sequencing and Gene Analysis
Lecture ONE: Foundation Course Genetics Tools of Human Molecular Genetics I.
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.
PCR quantitative en temps réel Lydie Pradel. PCR.
Gene Regulation: What it is, and how to detect it By Jordan, Jennifer, and Brian.
Enzyme names to learn 1.Reverse transcriptase 2.RNA polymerase 3.DNA helicase 4.DNA ligase 5.DNA polymerase 6.Restriction endonuclease A.Unwinds DNA helix.
Genomic DNA purification
The polymerase chain reaction (PCR) rapidly
DNA Replication DNA mRNA protein transcription translation replication Before each cell division the DNA must be replicated so each daughter cell can get.
By Moayed al Suleiman Suleiman al borican Ahmad al Ahmadi
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