1. Experimental Design May 2007 Hanne Jarmer

2. What is Experimental Design? Question Answer Data Analysis Experiments Often not the final answer!

3. What do we need? What is the question(s)? Which experiment(s) will give the answer? How many replicates do we need? The “correct” experimental design?! Several equally good designs Balancing Replicating

4. The Question What is a good question?... that can be answered by the use microarray experiments?

5. The Questions Why do obese children have diabetes more frequently? What is the connection to fitness/diet? ?

6. The Experiments 1.From question: Define your categories 2.Access to material? (living human brain?) 3.How many replications?

7. The Experiments Determine from where you’d get variation? Hopefully: Biology !! - array - spot - time - patient gender/age/other - dye - technician

8. The Experiments From question: Define your categories Sick healthy

9. The Experiments Access to material?... Mmmm, three of these... Human samples not always possible... Maybe rats instead?

10. The Experiments How many replications? Impossible to answer! Depends on how...... strong the biological signal is... much you’d like to be able to trust the result... you plan to analyze... much you can afford... Biological replications are always preferable!

11. The Experiments Variation... How do we get (only) what we want? THE KEY: BALANCING.. all other sources of variation SICK MENHEALTY MEN2 old, 6 young 3 day A, 3 day B 2 batch 1, 4 batch 2 Technician Y

12. DYE SWAP... is it really necessary? The non-linear normalization should take care of this... right?! This is mostly true,... but not always: “Technical” or “biological” dye swap? A biological replication always give you more !!

13. The Experiments Important: Pick conditions that:... will maximize the interesting biological signal Ex.: Wild-type bacteria TF-mutant... and... choose a growth medium where the TF is active!! TF List of genes?

14. The Experiments Important: Pick conditions that:... will maximize the interesting biological signal... while minimizing the overall difference Less optimal:Liver Brain

15. Basic Designs “Reference design” “Loop design” Stanford-type microarrays:

16. Basic Designs “Loop design” 2 samples from the same category

17. Please, take home... 1.Question How to save the world? 2.CategoriesA - B -C 3.Balance 4.Replication (biological)

18. Example: Why not AIDS? 1.Question: Why do some HIV- infected... not develop AIDS? 2.Categories: HIV-infected Patient type 0100 10 11 0 = no AIDS 1 = AIDS 0 = no HIV 1 = HIV NEVER AIDS

19. Example: Why not AIDS? Time Blood samples HIV AIDS 00 01 10 11 ~ 20 years HIV-infected Patient type 0100 10 11 0 = no AIDS 1 = AIDS 0 = no HIV 1 = HIV Determination of categories

20. Example: Why not AIDS? Balancing and replicating Variation can come from: Village, age, doctor,.... T H -cell extraction Relatively few “0” patients... - Pick corresponding “1” patients

21. Example: Why not AIDS? HIV-infected Patient type 0 = no AIDS 1 = AIDS 0 = no HIV 1 = HIV 01 11 00 10 3 P-values for each gene...

22. Example: Why not AIDS? The 2-way ANOVA results: 3 lists: - Patient-type difference - +/- HIV difference - The Interaction: “0”-patient genes that behave unexpectedly during HIV-infection

23. Example: Why not AIDS? -CCR5-mutation (no HIV) -The golden combination of HLAs (HLA-B57 in particular) -Something unknown !?

24. Please, take home... 1.Question How to save the world? 2.CategoriesA - B -C 3.Balance 4.Replication (biological)

25. Short Discussion A)Questions: 1)How will Treatment A or B or the combination of both affect the genes in the malaria parasite? 2)Will either affect the production of the “sticky” proteins responsible for the aggregation of the red blood cells?

26. Short Discussion B) How would you design this study? C) Which categories? No treatment Treatment A Treatment B The combination

27. Short Discussion - in groups The study is done using two-color microarrays and 4 biological replications D)Which samples would you label red and which would you label green? E)What will you hybridize with what? N-1A-1B-1AB-1 N-2A-2B-2AB-2 N-3A-3B-3AB-3 N-4A-4B-4AB-4 N-1A-1B-1AB-1 N-2A-2B-2AB-2 N-3A-3B-3AB-3 N-4A-4B-4AB-4

28. Short Discussion - in groups N-1 B-1 A-2 AB-2 N-3 B-3 A-4 AB-4 N-2 AB-3 A-3 B-4 AB-1 N-4 B-2 A-1

29. Short Discussion - in groups A)Can you come up with another interesting biological question? B)Which categories would you need? C) How would you balance this study?