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09/17/16 1
Micro-array study for gene
expression
09/17/16 2
History
cDNA microarrays have evolved from Southern blots, withcDNA microarrays have evolved from Southern blot...
09/17/16 3
Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The ...
09/17/16 4Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The W...
09/17/16 5Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The W...
09/17/16 6Department of Statistics, University of California, Berkeley, and
Hybing the Chip:
ARRAY HYBRIDIZATION
PROBE LAB...
09/17/16 7Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The W...
09/17/16 8
Schematic of probe preparation , hybridization,
scanning and immage analysis
09/17/16 9
09/17/16 10
Our Aim
• We intend to induce rupture of atherosclerotic plaque in
apo-e mice by injecting several drugs, whic...
09/17/16 11
DNA Sequence Synthesis
 We are going to design a genearray slide based onWe are going to design a genearray s...
09/17/16 12Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The ...
09/17/16 13
09/17/16 14
09/17/16 15
 We will order a probe to be made by a company that willWe will order a probe to be made by a company that wi...
09/17/16 16
• Drugs being used to
induce rupture of
atherosclerotic plaque
in apo-e mice
09/17/16 17
Drugs being used
• LNAME
• Adrenalin
• Cocaine
• Xanthine
• Xanthine Oxidase
• Interleukin-1 beta
• Methionine...
09/17/16 18
If we do not find
anything
Very pleasant, at least
we shalll find
something new
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Mol gen-1

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Mol gen-1

  1. 1. 09/17/16 1 Micro-array study for gene expression
  2. 2. 09/17/16 2 History cDNA microarrays have evolved from Southern blots, withcDNA microarrays have evolved from Southern blots, with clone libraries gridded out on nylon membrane filtersclone libraries gridded out on nylon membrane filters being an important and still widely used intermediate.being an important and still widely used intermediate. Things took off with the introduction of non-porous solidThings took off with the introduction of non-porous solid supports, such as glass - these permitted miniaturization -supports, such as glass - these permitted miniaturization - and fluorescence based detection. Currently, about 20,000and fluorescence based detection. Currently, about 20,000 cDNAs can be spotted onto a microscope slide.cDNAs can be spotted onto a microscope slide.
  3. 3. 09/17/16 3 Department of Statistics, University of California, Berkeley, and Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research, THE PROCESSTHE PROCESS Building the Chip: MASSIVE PCR PCR PURIFICATION and PREPARATION PREPARING SLIDES PRINTING Preparing RNA: CELL CULTURE AND HARVEST RNA ISOLATION cDNA PRODUCTION Hybing the Chip: POST PROCESSING ARRAY HYBRIDIZATION PROBE LABELING DATA ANALYSIS
  4. 4. 09/17/16 4Department of Statistics, University of California, Berkeley, and Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research, cDNA clones (probes) PCR product amplification purification printing microarray Hybridise target to microarray mRNA target) excitation laser 1laser 2 emission scanning analysis overlay images and normalise 0.1nl/spot
  5. 5. 09/17/16 5Department of Statistics, University of California, Berkeley, and Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research, Preparing RNA: RNA ISOLATION cDNA PRODUCTION Designing experiments to profile conditions/perturbations/ mutations and carefully controlled growth conditions RNA yield and purity are determined by system. PolyA isolation is preferable but total RNA is useable. Two RNA samples are hybridized/chip. Single strand synthesis or amplification of RNA can be performed. cDNA production includes incorporation of Aminoallyl-dUTP. Department of Statistics, University of California, Berkeley, and Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research, Preparing RNA: RNA ISOLATION cDNA PRODUCTION Designing experiments to profile conditions/perturbations/ mutations and carefully controlled growth conditions RNA yield and purity are determined by system. PolyA isolation is preferable but total RNA is useable. Two RNA samples are hybridized/chip. Single strand synthesis or amplification of RNA can be performed. cDNA production includes incorporation of Aminoallyl-dUTP. Department of Statistics, University of California, Berkeley, and Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research, Preparing RNA: RNA ISOLATION cDNA PRODUCTION Designing experiments to obtain conditions necessary to induce changes in the plaque to cause its rupture RNA yield and purity are determined by system. Single strand synthesis or amplification of RNA can be performed. OBTAINING THE TARGET TISSUE
  6. 6. 09/17/16 6Department of Statistics, University of California, Berkeley, and Hybing the Chip: ARRAY HYBRIDIZATION PROBE LABELING DATA ANALYSIS Cy3 and Cy5 RNA samples are simultaneously hybridized to chip. Hybs are performed for 5-12 hours and then chips are washed. Two RNA samples are labelled with Cy3 or Cy5 monofunctional dyes via a chemical coupling to AA-dUTP. Samples are purified using a PCR cleanup kit. Ratio measurements are determined via quantification of 532 nm and 635 nm emission values. Data are uploaded to the appropriate database where statistical and other analyses can then be performed.
  7. 7. 09/17/16 7Department of Statistics, University of California, Berkeley, and Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research, Biological Question Sample preparationMicroarray Life Cycle Data Analysis & Modeling Microarray Reaction Microarray Detection aken from Schena & Davis
  8. 8. 09/17/16 8 Schematic of probe preparation , hybridization, scanning and immage analysis
  9. 9. 09/17/16 9
  10. 10. 09/17/16 10 Our Aim • We intend to induce rupture of atherosclerotic plaque in apo-e mice by injecting several drugs, which we hope will cause rupture of the plaque by altering the hemodynamic system and raising the oxidative stress and alter the composition of the plaque. • After achieving this goal we will perform gene- array study on these specimens and will try to find out which genes are expressed more in plaques which are ruptured or are prone to rupture, by comparing our results at the histopathology lab which will define the structural details of such vulnerable plaques.
  11. 11. 09/17/16 11 DNA Sequence Synthesis  We are going to design a genearray slide based onWe are going to design a genearray slide based on oligonucleotide sequences that we will deriveoligonucleotide sequences that we will derive from the genes that we are interested in.from the genes that we are interested in.  To do this, we will proceed as follows:To do this, we will proceed as follows:  First we will gather the cDNA or mRNAFirst we will gather the cDNA or mRNA sequences of the desired genes from Entrez.sequences of the desired genes from Entrez.  Then we will pickup a sequence of genes from thatThen we will pickup a sequence of genes from that particular gene and by using Primer Premierparticular gene and by using Primer Premier program we will design a primer to use as aprogram we will design a primer to use as a hybridization probe.hybridization probe.
  12. 12. 09/17/16 12Department of Statistics, University of California, Berkeley, and Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
  13. 13. 09/17/16 13
  14. 14. 09/17/16 14
  15. 15. 09/17/16 15  We will order a probe to be made by a company that willWe will order a probe to be made by a company that will make that on a commercial basis.make that on a commercial basis.  The micro array-chip, we will make at the lab byThe micro array-chip, we will make at the lab by synthesizing cDNA from mRNA, that we isolated.synthesizing cDNA from mRNA, that we isolated.  We will obtain the tissue samples from the mice, accordingWe will obtain the tissue samples from the mice, according to our study design, we will purify the mRNA accordingto our study design, we will purify the mRNA according the specific protocol.the specific protocol.  mRNA from the target will be converted into cDNA, andmRNA from the target will be converted into cDNA, and allowed to hybridiz on the microarray chip that weallowed to hybridiz on the microarray chip that we designeddesigned  The emission of colors will be captured by the scanner,The emission of colors will be captured by the scanner, and the results will be analysed depending upon theirand the results will be analysed depending upon their intensity and presence and absence of colorintensity and presence and absence of color
  16. 16. 09/17/16 16 • Drugs being used to induce rupture of atherosclerotic plaque in apo-e mice
  17. 17. 09/17/16 17 Drugs being used • LNAME • Adrenalin • Cocaine • Xanthine • Xanthine Oxidase • Interleukin-1 beta • Methionine • Buthionine Sulfoximine
  18. 18. 09/17/16 18 If we do not find anything Very pleasant, at least we shalll find something new

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