1 / 21

Large Scale Gene Expression with DNA Microarrays

Large Scale Gene Expression with DNA Microarrays. Vermont Genetics Network Microarray Outreach Program. Vermont Genetics Network (VGN) Founded at the University of Vermont in 2001 through an NIH BRIN grant and renewed in 2005 through an NIH INBRE grant Purpose:

duncan
Download Presentation

Large Scale Gene Expression with DNA Microarrays

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Large Scale Gene Expression with DNA Microarrays Vermont Genetics Network Microarray Outreach Program

  2. Vermont Genetics Network (VGN) • Founded at the University of Vermont in 2001 • through an NIH BRIN grant and renewed in 2005 through an NIH INBRE grant • Purpose: • Encourage biomedical research in Vermont • Create a “network” of researchers and students • Give outreach lectures to 4-year institutions • Provide research grants to faculty and students • Mentoring for students interested in research

  3. VGN Microarray Outreach Program • Develop microarray outreach module. • Introduce microarray technology to VT colleges. • Team of scientists to serve as instructors • Ahmad Chaudhry, Tim Hunter, Pat Reed • Scott Tighe, Janet Murray

  4. Microarray Experiment • The effect of a chemical dimethyl sulfoxide (DMSO) • on gene expression in yeast • DMSO is an environmental contaminant from the paper industry and from pesticides • Grow the yeast and treat one group with plain water • (control group) and the other group with 10% DMSO • Isolate RNA from the yeast grown in two different conditions, prepare target from it and use it on microarrays to see changes in gene expression

  5. Expressed Genes = mRNA DNA (genes) messenger RNA Protein (effector molecules)

  6. What are Microarrays? • Microarrays are simply small glass or silicon slides upon the surface of which are arrayed thousands of genes (usually between 500-20,000) • Via a conventional DNA hybridization process, the level of expression/activity of genes is measured • Data are read using laser-activated fluorescence readers • The process is “ultra-high throughput”

  7. Why use Microarrays? • What genes are Present/Absent in a cell? • What genes are Present/Absent in the experiment vs. control? • Which genes have increased/decreased expression in experiment vs. control? • Which genes have biological significance?

  8. Why analyze so many genes? • Just because we sequenced a genome doesn’t mean we know anything about the genes. Thousands of genes remain without an assigned function. • Patterns/clusters of expression are more predictive than looking at one or two prognostic markers – can figure out new pathways

  9. Experimental Design • Choice of reference: Common (non-biologically relevant) reference, or paired samples? • Number of replicates: How many are needed? (How many are affordable?). Are the replicate results going to be averaged or treated independently? Is this a “fishing expedition” or a hypothesis-based experiment?

  10. The steps of a microarray experiment:

  11. mRNA cDNA cDNA cRNA Creating Targets Reverse Transcriptase 1st Strand 2nd Strand in vitro transcription

  12. RNA-DNA Hybridization Targets (RNA) probe sets on chip (DNA) (25 base oligonucleotides of known sequence)

  13. Non-Hybridized Targets are Washed Away Targets (fluorescently tagged) “probe sets” (oligos) Non-bound ones are washed away

  14. Why Use Yeast (S. cerevisiae)?? • easily manipulated in the laboratory • simple eukaryote, unicellular • rapid growth (doubling 1.5 - 2.5 hours) • non-pathogenic • stable haploid and diploid states • complete genome sequenced

  15. E. coli ~ 1 x 3 m Yeast ~ 5 m dia. Human ~ 1.7 m 1 chromosome 4 x 10 6 bp 16 chromosomes 12 x 10 6 bp 23 chromosomes 3.3 x 10 9 bp ~ 30, 000 genes ~ 4,377 genes ~ 5,726 genes

  16. Yeast Life cycle

  17. S. cerevisiae Genome • DNA ~ 60% A + T • 16 chromosomes (haploid) • chromosomes have centromeres and telomeres • also have mitochondrial genome • plasmid: 6 kb, 60-100 copies/cell • entire genome has been sequenced

  18. Yeast Genetic Nomenclature • genes names are 3-letters + a number • genes names are derived from phenotype of the mutant, i.e. ste 3 mutants have sterile phenotype • gene names are written in italics or underlined • wild type alleles: Uppercase STE 3 • mutant alleles: lowercase ste 3 Yeast strain : NRRL Y12362

  19. Growth and Metabolism + O2 / no O2 CARBON + NITROGEN + BIOTIN + MINERALS Prototroph: requires no additional nutrients (can grow in minimal media) Auxotroph: requires additional nutrients from the environment or media NRRL Y12362 is auxotrophic for leucine, tryptophan, uracil, adenine, and histidine YPD is a rich media; therefore, NRRL Y12362 can get these nutrients from the environment

  20. Changes in gene expression? Which genes are up regulated? Which genes are down regulated? What do the results say about yeast biology? What parallels (if any) can we make to human biology? untreated versus DMSO treated Yeast Microarray Experiment

More Related