Microarray experiment question..Please help?

Question

You are interested in understanding how the brain works, and are using the fruit fly Drosphila as a model system to study brain development. You perform microarray analysis to try to determine genes expressed in the fly brain. For your microarray experiment, you first prepare cDNA from fly brains and label it with a red fluorchrome. Then you isolate cDNA from whole flies and label it with a green fluorchrome. Next you hybridize these cDNA populations to a microarray containing the Drosphila genes. From this, you obtain a list of genes that are specifically enriched in the brain. (those that show up as a red spot on the microarray). You are dissappointed bc your favorite fly gene, tubby, does not appear on this list, even though you have repeated the miroarray experiment 10 times and did not encounter any technical difficulties. The reason you thought tubby would appear on this list is that you believe tubby is important for brain development, since flies mutant in tubby have no brains.

Not to be discouraged, you perform in situ analysis using the tubby DNA as a probe, and see that it is expressed at high levels in the fly brain of normal flies but not expressed in animals lacking the tubby gene. Why do you think tubby did not show up as a gene specifically enriched in the brain in your microarray experiment? (you did the experiment corrrectly)

Answer 1

It would appear that though Tubby is important for brain development, that it is not expressed only in the brain, but in the rest of the fly body as well, and therefore should turn up yellow in the 10 results.

Answer 2

Microarrays in general are not a great tool for specific answers like you are looking for. Multiplex hybs are severely complicated things with an insane number of different kinetic reactions between all the target strands and all the probe strands. That said, there could be a ton of reasons why this is happening.
1. It really is happening but the microarray isn't detecting it. This could be due to the sample prep, missing the tissue of interest, or the MA just being annotated wrong. It may be that the gene you think is in that spot is something else completely. Check to see if the contents of the spot have been sequence verified.
2. There could be a dye labeling bias. You are probably using Cy3 and Cy5 as your fluorophores. If you are using direct incorporation of dye-fluor conjugates, this could be a factor. Cy5 doesn't incorporate as well due to it's larger size.
3. You mention tubby being vital to development. Try taking samples from embryos or other timepoints you think relevant.
4. tubby may not be involved in neural tissue development. Perhaps it is dealing with formation of other structures in the head.
5. The mutant gene may not hyb well to the probe on the chip due to mismatches.
6. Try the experiment versus wild type. The control can make a huge difference as a common reference. Remember, MA's are just relative measures, not absolute!
7. Are the whole fly samples from flies with no brains? Maybe the absense of tubby in the flies with no brains is what you are looking for!

I could probably come up with a few more reasons, but you get the point. It's been a while since I have taken Genetics but if this is a homework or test quesiton, you are probably better equipped to deal with the actual genetics of the problem. :-P