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Microarrays Other sites that you may need quick access to: Go to the website: http://genome-www5.stanford.edu/MicroArray/SMD/index.shtml This site has Microarray experimental data posted for anyone to analyze. There are a lot of experiments readily available at this point, and such databases will likely become more common. There are currently over 18,300 experiments (up from 132 in 2001, 8899 in fall of 2005, 13,000 in 2007), mostly from Arabidopsis, yeast, and humans. We will be doing some analysis of experiment 3610. You will be doing a Search and under the Search by experiments heading you will have a series of options. Go to Select Organism and choose Arabidopsis thaliana. Click Display Data to retrieve the Arabidopsis experiments (there are ~1100). Re-sort the data by ExptID and find experiment 3610. 1) Click on the View Array Details icon for this experiment. What is experiment 3610 comparing? What is the Channel 1 probe? Channel 2 probe? What does etiolated mean? Go back to the experiment list and click on the View and Sort Array Data icon for experiment 3610 . Choose sort by log(base 2)of R/G Normalized Ratio(Mean) and descending order, for display choose Description in the far right box. Uncheck all filters for all analyses from now on, unless instructed otherwise. 2) How is the data going to be sorted? (think about what R/G and descending mean) 3) Click Display and look at the top 10-15 ESTs. What metabolic process do the ESTs with the highest R/G ratios have in common? In which condition are they most highly expressed? Was this expected? Look at the eleventh ranked EST. 4) What is the log(base 2) R/G ratio? What does the EST encode? Click Zoom. This will give you a look at the actual spot on the microarray. (If you click the spot itself, you can download an image of the entire microarray with your spot of interest in a yellow box.) Use the Accession Number to look up the sequence of this EST in GENBANK. Remember that this is the sequence of an EST and does NOT contain the entire gene sequence, so it will be short. 5) What is the accession number? 6) What would you do to check if there are any similar sequences currently in GENBANK? Do it. 7) List three other organisms that contain a sequence with significant similarity to this one. What proteins do these sequences encode and why do you think the similarity is significant?
8) In what metabolic process is this protein likely to be involved in these organisms? Go back to the data spot in the microarray database and view the expression history of this spot in the other Arabidopsis experiments. 9) What can we accomplish or what might we learn by looking at the expression in other experiments?
10) Taking a log(base 2) R/G ratio of ±2.0 (a 4-fold difference in gene expression) to be possibly significant, are there any other bars in the histogram (other than the current spot) that could indicate a significant change in expression? For how many experiments is there a significant difference in gene expression?
11) Pick the experiment that shows the largest negative ratio for this EST and write down the experiment number, name, and log2 Ratio . What tissues/growth conditions were being tested? Based on your answer to question 8, does the difference in expression for this gene make sense? Why or why not?
12) Zoom in on the spot from the new experiment, is it more red or less red than the spot in experiment 3610? Why?
Go back to experiment 3610 and run the same analysis (described after question 1 above), except this time sort in ascending order. Keep the other parameters the same. 13) Do the ESTs with the highest G/R ratios have any metabolic process in common? Why do you think this is so?
Look at the 23rd ranked EST. What does the description suggest that the gene from which this EST is derived does? 14) List the Names (which are actually letters and numbers) of the 22nd-26th ranked ESTs. Zoom in on each spot one at a time to look at their colors. What seems wrong? Now go back and rerun the analysis using filters four and five. Keep the other parameters the same. 15) List the Names of the 21st-25th ranked ESTs. What happened to the former 23rd ranked EST? Why?
16) Suppose you have a favorite gene and would like to see if it changes expression significantly under any of the conditions found in the database. Describe how you would find this information.
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