1. D N A Microarrays 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

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

14. 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

15. Fluorescent marking of mRNA occurs through reverse transcription

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

17. 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/

18. Hybridization of two lines to a chip

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

20. 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

21. Comparing fluorescence levels 100400 300400 300 100400 40.75 0.331 Green (normal) Fluorescence Red (tumor) Fluorescence Ratio Red to Green

22. AB DC Gene AGene BGene CGene D Red (tumor) Fluorescence 400200100400 Green (Normal) Fluorescence 100200300400 Ratio Red to Green 410.331

23. Gene AGene BGene CGene D Sample 1410.331 Sample 2140.331 Sample 340.330.752 Sample 41440.33 Sample 50.330.524 Sample 61114 Sample 70.33144 Compile ratios from many sample sets

24. Gene AGene BGene CGene D Sample 10.65000.333-0.333 Sample 2.6-0.450.650 Sample 30.1000.500-0.7500.100 Sample 400.5000.3450.100 Sample 5-0.550-0.300-0.335-0.100 Sample 60.5000.300-0.1000 Sample 70.3000.110-0.1000.050 Transform each ratio by Log 2

25. Assign each box a score based on this relative expression level x10 1:1 RepressedInduced

26. Gene AGene BGene CGene D Sample 10.65000.333-0.333 Sample 2.6-0.450.650 Sample 30.1000.500-0.7500.100 Sample 400.5000.3450.100 Sample 5-0.550-0.300-0.335-0.100 Sample 60.5000.300-0.1000 Sample 70.3000.110-0.1000.050

28. 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

29. We can then compare scores between each gene Gene AGene BGene C Gene A10.5-0.3 Gene B-0.510.3 Gene C0.5 1 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)

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

31. Gene AGene BGene CGene D Sample 1 0.65000.333-0.333 Sample 2.6-0.450.650 Sample 3 0.1000.500-0.7500.100 Sample 4 00.5000.3450.100 Sample 5 -0.550-0.300-0.335-0.100 Sample 6 0.5000.300-0.1000 Sample 7 0.3000.110-0.1000.050

32. Gene AGene BGene CGene D Sample 1 0.65000.333-0.333 Sample 2.6-0.450.650 Sample 3 0.1000.500-0.7500.100 Sample 4 00.5000.3450.100 Sample 5 -0.550-0.300-0.335-0.100 Sample 6 0.5000.300-0.1000 Sample 7 0.3000.110-0.1000.050

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

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

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

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

38. 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

39. 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_conten t&task=view&id=70&Itemid=20

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

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

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

43. 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/GnpSN P/data_summary.php

44. 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 Northern blot shows expression of CAB1, HAT4, ROC1, and human AChR Schena et al.

45. Table 1 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 Schena et al.

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

47. Figure 1: C and D 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 Schena et al.

48. Figure 1: E and F 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 Schena et al.

49. 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.

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

51. Surprise! HAT4 phenotype: elongated hypocotyls, early flowering, poor germination, altered pigmentation Only 1 gene found with differential expression Image retrieved from http://en.wikipedia.org/wiki/File:Salix_ scouleriana.seed.jpg Image retrieved from http://www.1up.com/do/my1Up?p ublicUserId=5685007

52. Conclusions Microarrays are really cool. Expressed Sequence Tags=EST 20,274 ESTs for A. thaliana Uses? Linking diseases and treatments with human gene sequences

53. Borevitz and Ecker. (2004) I assume you can read that? ESTs for A. thaliana: 190732 ESTs for H. sapiens: 5427257

54. Array Express websearch

57. Questions? I recently read a paper that linked the composition of the human microbiome to obesity in twins. Along these lines, do you think that micro arrays could be used to assay the levels of gene expression of the human microbiome to study pathogens or other problems that may be due to microbes?

58. I recently read about a new type of microarray technique that is based on electrostatics. The article discussed how it is much more cost efficient, does not need complex chemical labels, and is more sensitive than traditional techniques. The article made it seem like this was going to revolutionize diagnostic and personalized medicine. I was just curious if you had come across anything else about this technique and whether it was in fact being used in labs or clinics?

59. Acknowledgements Ahna Skop University of Wisconsin-Madison The attentive audience Thank you for listening