1. 1 Experimental Design EPP 245 Statistical Analysis of Laboratory Data

2. October 4, 2007EPP 245 Statistical Analysis of Laboratory Data 2 Basic Principles of Experimental Investigation Sequential Experimentation Comparison Manipulation Randomization Blocking Simultaneous variation of factors Main effects and interactions Sources of variability Issues with two-color arrays

3. October 4, 2007EPP 245 Statistical Analysis of Laboratory Data 3 Sequential Experimentation No single experiment is definitive Each experimental result suggests other experiments Scientific investigation is iterative. “No experiment can do everything; every experiment should do something,” George Box.

4. October 4, 2007EPP 245 Statistical Analysis of Laboratory Data 4 Plan Experiment Perform Experiment Analyze Data from Experiment

5. October 4, 2007EPP 245 Statistical Analysis of Laboratory Data 5 Comparison Usually absolute data are meaningless, only comparative data are meaningful The level of mRNA in a sample of liver cells is not meaningful The comparison of the mRNA levels in samples from normal and diseased liver cells is meaningful

6. October 4, 2007EPP 245 Statistical Analysis of Laboratory Data 6 Internal vs. External Comparison Comparison of an experimental results with historical results is likely to mislead Many factors that can influence results other than the intended treatment Best to include controls or other comparisons in each experiment

7. October 4, 2007EPP 245 Statistical Analysis of Laboratory Data 7 Manipulation Different experimental conditions need to be imposed by the experimenters, not just observed, if at all possible The rate of complications in cardiac artery bypass graft surgery may depend on many factors which are not controlled and may be hard to measure

8. October 4, 2007EPP 245 Statistical Analysis of Laboratory Data 8

9. October 4, 2007EPP 245 Statistical Analysis of Laboratory Data 9 Randomization Randomization limits the difference between groups that are due to irrelevant factors Such differences will still exist, but can be quantified by analyzing the randomization This is a method of controlling for unknown confounding factors

10. October 4, 2007EPP 245 Statistical Analysis of Laboratory Data 10 Suppose that 50% of a patient population is female A sample of 100 patients will not generally have exactly 50% females Numbers of females between 40 and 60 would not be surprising In two groups of 100, the disparity between the number of females in the two groups can be as big as 20% simply by chance This also holds for factors we don’t know about

11. October 4, 2007EPP 245 Statistical Analysis of Laboratory Data 11 Randomization does not exactly balance against any specific factor To do that one should employ blocking Instead it provides a way of quantifying possible imbalance even of unknown factors Randomization even provides an automatic method of analysis that depends on the design and randomization technique.

12. October 4, 2007EPP 245 Statistical Analysis of Laboratory Data 12 The Farmer from Whidbey Island Visited the University of Washington with a Whalebone water douser 10 Dixie cups, 5 with water, 5 empty, covered with plywood If he gets all 10 right, is chance a reasonable explanation?

13. October 4, 2007EPP 245 Statistical Analysis of Laboratory Data 13 The randomness is produced by the process of randomly choosing which 5 of the 10 are to contain water There are no other assumptions

14. October 4, 2007EPP 245 Statistical Analysis of Laboratory Data 14 If the randomization had been to flip a coin for each of the 10 cups, then the probability of getting all 10 right by chance is different There are 2 10 = 1024 ways for the randomization to come out, only one of which is right, so the chance is 1/1024 =.001 The method of randomization matters

15. October 4, 2007EPP 245 Statistical Analysis of Laboratory Data 15 Randomization Inference 20 tomato plants are divided 10 groups of 2 placed next to each other in the greenhouse In each group of 2, one is chosen to receive fertilizer A and one to receive fertilizer B The yield of each plant is measured

16. October 4, 2007EPP 245 Statistical Analysis of Laboratory Data 16 12345678910 A13282109143107669510888133 B14088112142118649811393136 diff86311-23553

17. October 4, 2007EPP 245 Statistical Analysis of Laboratory Data 17 Average difference is 4.1 Could this have happened by chance? Is it statistically significant? If A and B do not differ in their effects (null hypothesis is true), then the plants’ yields would have been the same either whether A or B is applied The difference would be the negative of what it was if the coin flip had come out the other way

18. October 4, 2007EPP 245 Statistical Analysis of Laboratory Data 18 In pair 1, the yields were 132 and 140. The difference was 8, but it could have been -8 With 10 coin flips, there are 2 10 = 1024 possible outcomes of + or – on the difference These outcomes are possible outcomes from our action of randomization, and carry no assumptions

19. October 4, 2007EPP 245 Statistical Analysis of Laboratory Data 19 Of the 1024 possible outcomes that are all equally likely under the null hypothesis, only 3 had greater values of the average difference, and only four (including the one observed) had the same value of the average difference The likelihood of this happening by chance is [3+4/2]/1024 =.005 This does not depend on any assumptions other than that the randomization was correctly done

20. October 4, 2007EPP 245 Statistical Analysis of Laboratory Data 20 12345678910 A13282109143107669510888133 B14088112142118649811393136 diff86311-23553

21. October 4, 2007EPP 245 Statistical Analysis of Laboratory Data 21

22. October 4, 2007EPP 245 Statistical Analysis of Laboratory Data 22 Randomization and in practice Whenever there is a choice, it should be made using a formal randomization procedure, such as Excel’s rand() function. This protects against unexpected sources of variability such as day, time of day, operator, reagent, etc.

23. October 4, 2007EPP 245 Statistical Analysis of Laboratory Data 23 Pair NumberFirst Sample Treatment 1A or B? 2 3 4 5 6 7 8 9 10A or B?

24. October 4, 2007EPP 245 Statistical Analysis of Laboratory Data 24 Pair Num First Sample Treatment random number 1A or B?0.871413 2A or B?0.786036 3A or B?0.889785 4A or B?0.081120 5A or B?0.297614 6A or B?0.540483 7A or B?0.824491 8A or B?0.624133 9A or B?0.913187 10A or B?0.001599

25. October 4, 2007EPP 245 Statistical Analysis of Laboratory Data 25 =rand() in first cell Copy down the column Highlight entire column ^c (Edit/Copy) Edit/Paste Special/Values This fixes the random numbers so they do not recompute each time =IF(C3<0.5,"A","B") goes in cell C2, then copy down the column

26. October 4, 2007EPP 245 Statistical Analysis of Laboratory Data 26 Plant Pair First Plant Treatment random number 1B0.871413 2B0.786036 3B0.889785 4A0.081120 5A0.297614 6B0.540483 7B0.824491 8B0.624133 9B0.913187 10A0.001599

27. October 4, 2007EPP 245 Statistical Analysis of Laboratory Data 27 To randomize run order, insert a column of random numbers, then sort on that column More complex randomizations require more care, but this is quite important and worth the trouble Randomization can be done in Excel, R, or anything that can generate random numbers

28. October 4, 2007EPP 245 Statistical Analysis of Laboratory Data 28 Blocking If some factor may interfere with the experimental results by introducing unwanted variability, one can block on that factor In agricultural field trials, soil and other location effects can be important, so plots of land are subdivided to test the different treatments. This is the origin of the idea

29. October 4, 2007EPP 245 Statistical Analysis of Laboratory Data 29 If we are comparing treatments, the more alike the units are to which we apply the treatment, the more sensitive the comparison. Within blocks, treatments should be randomized Paired comparisons are a simple example of randomized blocks as in the tomato plant example

30. October 4, 2007EPP 245 Statistical Analysis of Laboratory Data 30 Simultaneous Variation of Factors The simplistic idea of “science” is to hold all things constant except for one experimental factor, and then vary that one thing This misses interactions and can be statistically inefficient Multi-factor designs are often preferable

31. October 4, 2007EPP 245 Statistical Analysis of Laboratory Data 31 Interactions Sometimes (often) the effect of one variable depends on the levels of another one This cannot be detected by one-factor-at- a-time experiments These interactions are often scientifically the most important

32. October 4, 2007EPP 245 Statistical Analysis of Laboratory Data 32 Experiment 1. I compare the room before and after I drop a liter of gasoline on the desk. Result: we all leave because of the odor.

33. October 4, 2007EPP 245 Statistical Analysis of Laboratory Data 33 Experiment 1. I compare the room before and after I drop a liter of gasoline on the desk. Result: we all leave because of the odor. Experiment 2. I compare the room before and after I drop a lighted match on the desk. Result: no effect other than a small scorch mark.

34. October 4, 2007EPP 245 Statistical Analysis of Laboratory Data 34 Experiment 1. I compare the room before and after I drop a liter of gasoline on the desk. Result: we all leave because of the odor. Experiment 2. I compare the room before and after I drop a lighted match on the desk. Result: no effect other than a small scorch mark. Experiment 3. I compare all four of ±gasoline and ±match. Result: we are all killed. Large Interaction effect

35. October 4, 2007EPP 245 Statistical Analysis of Laboratory Data 35 Statistical Efficiency Suppose I compare the expression of a gene in a cell culture of either keratinocytes or fibroblasts, confluent and nonconfluent, with or without a possibly stimulating hormone, with 2 cultures in each condition, requiring 16 cultures

36. October 4, 2007EPP 245 Statistical Analysis of Laboratory Data 36 I can compare the cell types as an average of 8 cultures vs. 8 cultures I can do the same with the other two factors This is more efficient than 3 separate experiments with the same controls, using 48 cultures Can also see if cell types react differently to hormone application (interaction)

37. October 4, 2007EPP 245 Statistical Analysis of Laboratory Data 37 Fractional Factorial Designs When it is not known which of many factors may be important, fractional factorial designs can be helpful With 7 factors each at 2 levels, ordinarily this would require 2 7 = 128 experiments This can be done in 8 experiments instead

38. October 4, 2007EPP 245 Statistical Analysis of Laboratory Data 38 F1F2F3F4F5F6F7 1HHHHHHH 2HHLHLLL 3HLHLHLL 4HLLLLHH 5LHHLLHL 6LHLLHLH 7LLHHLLH 8LLLHHHL

39. October 4, 2007EPP 245 Statistical Analysis of Laboratory Data 39 F1F2F3F4F5F6F7 1HHHHHHH 2HHLHLLL 3HLHLHLL 4HLLLLHH 5LHHLLHL 6LHLLHLH 7LLHHLLH 8LLLHHHL

40. October 4, 2007EPP 245 Statistical Analysis of Laboratory Data 40 F1F2F3F4F5F6F7 1HHHHHHH 2HHLHLLL 3HLHLHLL 4HLLLLHH 5LHHLLHL 6LHLLHLH 7LLHHLLH 8LLLHHHL

41. October 4, 2007EPP 245 Statistical Analysis of Laboratory Data 41 F1F2F3F4F5F6F7 1HHHHHHH 2HHLHLLL 3HLHLHLL 4HLLLLHH 5LHHLLHL 6LHLLHLH 7LLHHLLH 8LLLHHHL

42. October 4, 2007EPP 245 Statistical Analysis of Laboratory Data 42 Main Effects and Interactions Factors Cell Type (C), State (S), Hormone (H) Response is expression of a gene The main effect C of cell type is the difference in average gene expression level between cell types

43. October 4, 2007EPP 245 Statistical Analysis of Laboratory Data 43 For the interaction between cell type and state, compute the difference in average gene expression between cell types separately for confluent and nonconfluent cultures. The difference of these differences is the interaction. The three-way interaction CSH is the difference in the two way interactions with and without the hormone stimulant.

44. October 4, 2007EPP 245 Statistical Analysis of Laboratory Data 44 Sources of Variability in Laboratory Analysis Intentional sources of variability are treatments and blocks There are many other sources of variability Biological variability between organisms or within an organism Technical variability of procedures like RNA extraction, labeling, hybridization, chips, etc.

45. October 4, 2007EPP 245 Statistical Analysis of Laboratory Data 45 Replication Almost always, biological variability is larger than technical variability, so most replicates should be biologically different, not just replicate analyses of the same samples (technical replicates) However, this can depend on the cost of the experiment vs. the cost of the sample 2D gels are so variable replication is required

46. October 4, 2007EPP 245 Statistical Analysis of Laboratory Data 46 Quality Control It is usually a good idea to identify factors that contribute to unwanted variability A study can be done in a given lab that examines the effects of day, time of day, operator, reagents, etc. This is almost always useful in starting with a new technology or in a new lab

47. October 4, 2007EPP 245 Statistical Analysis of Laboratory Data 47 Possible QC Design Possible factors: day, time of day, operator, reagent batch At two levels each, this is 16 experiments to be done over two days, with 4 each in morning and afternoon, with two operators and two reagent batches Analysis determines contributions to overall variability from each factor

48. October 4, 2007EPP 245 Statistical Analysis of Laboratory Data 48 References Statistics for Experimenters, Box, Hunter, and Hunter, John Wiley

49. October 4, 2007EPP 245 Statistical Analysis of Laboratory Data 49 Exercise You have a clinical study in which 10 patients will either get the standard treatment or a new treatment Randomize which 5 of the 10 get the new treatment so that all possible combinations can result. Use Excel or another formal randomization method. Randomize so that in each pair of pair of patients entered by date, one has the standard and one the new treatment. What are the advantages of each method? Why is randomization important?