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D N A M i cr o a rr a ys

D N A M i cr o a rr a ys. Eric Nickels. Sam Trammell. What is the need for Microarrays?. Northern blots for gene expression.

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D N A M i cr o a rr a ys

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  1. D N A Microarrays Eric Nickels Sam Trammell

  2. What is the need for Microarrays?

  3. Northern blots for gene expression B. WITEK-ZAWADA, A. KOJ. 2003. REGULATION OF EXPRESSION OF STROMYELYSIN-1 BY PROINFLAMMATORY CYTOKINES IN MOUSE BRAIN ASTROCYTES. Accessed from http://www.jpp.krakow.pl/journal/archive/1203/articles/02_article.html

  4. Expanding our view of the cell Northern blotting is limited in scope Often we need to see expression levels of a wide range of genes on wide range of cell types Provides a systematic manner to examine global gene expression patterns

  5. Scale up to microarrays Image from http://www.molecularstation.com/molecular-biology-images/508-microarray-pictures/68-affymetrix-microarray.html?size=big

  6. Microarrays enable exploratory research “A DNA microarray is an ordered array of nucleic acids, proteins, small molecules, that enables parallel analysis of complex biochemical samples.” -Schena et al. (Science 270, 467-470, 1995)

  7. Creating the Gene Chip We must attach ssDNA or RNA oligonucleotides to a support surface Covalent attachment strategies Electrostatic adsorption strategies

  8. Belosludtsev et al 2000. DNA microarray based on non-covalent oligonucleotide attachment and hybridization in two dimensions. Analytical Biochemistry

  9. SpotBot

  10. Customizable chips

  11. Automated, large scale chip production http://www.hhmi.org/biointeractive/media/gene_chips-lg.mov

  12. Comparing Relative mRNA expression Allows us to simultaneously analyze differential expression of genes between cell lines Used to determine which genes are active and which are repressed in cancer cell lines

  13. Fluorescent marking of mRNA occurs through reverse transcription

  14. Image from http://cswww.essex.ac.uk/staff/W.Langdon/genechip/

  15. Stringency of binding is controlled Changing the salt/buffer concentration and the temperature alters the binding stringency Low temperature/high Salt concentration yields low stringency High stringency means only perfect matches anneal; lower stringency allows for some level of single base differences http://cswww.essex.ac.uk/staff/W.Langdon/genechip/

  16. Hybridization of two lines to a chip

  17. Reading the chip Hybridization is quantized through fluorescent adsorption detected by a microarray chip scanner http://www.moleculardevices.com/pages/instruments/gn_genepix4000.html

  18. Statistical analysis of fluorescence We must measure the ratio of fluorescence between the two dyes Gain information of the relative level of expression compared to a standard cell

  19. Comparing fluorescence levels Green (normal) Fluorescence Red(tumor) Fluorescence Ratio Red to Green

  20. A B C D

  21. Compile ratios from many sample sets

  22. Transform each ratio by Log2

  23. Assign each box a score based on this relative expression level x10 1:1 x10 Repressed Induced

  24. Determining similarities in gene expression requires normalizing the data To do this, we take the mean of each sample and divide by its standard deviation

  25. We can then compare scores between each gene A positive score indicates similarity in expression A score of zero indicates no similarity in expression A negative score means expression is opposite (when one is induced, the other is repressed)

  26. Hierarchical clustering is used to create a dendrogram Gene A Gene C Gene B

  27. Shyamsundar et al. 2005. A DNA microarray survey of gene expression in normal human tissues. Genome Biology.

  28. Eisen et al. 1993. Cluster analysis and display of genome-wide expression patterns. PNAS.

  29. Statistical programs exist to run through thousands of lines of data

  30. Microarrays have diverse uses in research Experimental uses of microarrays goes beyond comparative gene expression. DNA Diagnostics and detection SNP Genotyping MicroRNA profiling

  31. ChIP on Chip analysis A combined technique allowing researchers to examine transcriptional regulators and their control of gene expression First a transcription factor is bound by an antibody and precipitated, then the annealed DNA strand is analyzed through a microrarray

  32. Protein microarrays and antibody microarrays exist as well Both detect protein expression Antibody microarrays are essentially the same as ELISA Image from http://newenglandbiolabs.de/en/index.php?option=com_content&task=view&id=70&Itemid=20

  33. Tissue microarrays offer a clinical diagnostic tool http://en.wikipedia.org/wiki/File:Rob7_melanoma.jpg

  34. Quantitative Monitoring of Gene Expression Patterns with a Complementary DNA Microarray Schena, M., Shalon, D., Davis, R., and Brown, P. (1995)

  35. Main Method Used What do you think it is? ? Microarray!!!!!!!

  36. Model Species Used Arabidopsis thaliana Smallest genome of any higher eurkaryote At time of paper, forty-five cloned Arabidopsis thaliana Easy to store Easy to obtain mutants Taken from http://urgi.versailles.inra.fr/projects/GnpSNP/data_summary.php

  37. Figure 1 and 2 Color bars were callibrated with signals from concentrations of human AChR mRNA. Numbers and letters correspond to positions of each cDNA Figure 1 Figure 2 Schena et al. Northern blot shows expression of CAB1, HAT4, ROC1, and human AChR Schena et al.

  38. Table 1 Schena et al. These are the positions of the significant genes hybridized in the study a1,2 AChR Human AChR b1,2 CABI Chlorophyll a/b binding c11,12 rGR Rat Glucocortoid receptor h11,12 TRP4 Yeast tryptophan biosynthesis

  39. Figure 1: A and B Schena et al. A: No hybridization between cDNA and mRNA for rat glucocorticoid receptor or yeast TRP4 targets at highest sensitivity B: Allowed for a comparison The two scans differed in sensitivity Result: Differential expression levels between the 45 genes tested

  40. Figure 1: C and D Schena et al. One array scanned for either fluorescein-labeled cDNA (wt) or lissamine-labeled cDNA (HAT4-transgenic plant) Intense expression of HAT4 in transgenic plant 50-fold elevation for HAT4

  41. Figure 1: E and F Schena et al. Fluorescein-labeled cDNA from root tissue (E) Lissamine-labeled cDNA from leaf tissue (F) mRNA from light-regulated CAB1 gene was ~500 fold 26 other genes differed in expression by more than a factor of 5

  42. Figure 2 No differential expression between wt and transgenic for CAB1 Expected differential expression between wt and transgenic for HAT4 No differential expression between wt and transgenic for ROC1 Schena et al.

  43. Table 2: Comparison between microarrary and Northern blot Schena et al.

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