Hybridization Preworkshop Assignment. 

    This assignment is recommended IN ADDITION to the data assignment (see website) for Hybridization workshop participants.  You should print out this set of questions and try to answer them before coming to the workshop; you can then add to your answers as you accumulate more direct experience.  We will discuss certain questions at particular points during the workshop.  The material that you need to answer questions 2-8 can be found in the article by DeRisi et al. referred to below.  We will try to send you a pdf of this article, but provide the reference in case you have trouble with the pdf file.   It is also assigned for the Data workshop but different aspects are highlighted for each workshop:

DeRisi, J. , Iyer, V, and Brown, P. O.  Exploring the metabolic and genetic control of gene expression on a global scale.  Science 278:680-686 (1997). 

1. Please answer the following with regard to your plans to use microarrays in an undergraduate class.
   1. How will microarray use relate to the overall goals of your class?

2. What concerns do you have about using this type of experiment in your class?

3. If you have chosen a type of microarray experiment that you would like your class to perform, what kind?

4. How much time are you planning to devote to microarray experiments?  To microarray data analysis?

5. What contingencies do you have in mind for problems that may arise with your microarray experiments?

2. We will be preparing total RNA and then we will copy the mRNA (a small minority of the RNA molecules present, about 1-3%) into cDNA. 
   1. Why won’t the noise from the other RNAs drown out the signal from the

mRNA?  (Hint: we will be using a short molecule of oligo dT (16 T nucleotides in a row) as the primer for reverse transcription, so you may want to consider how eukaryotic mRNAs are processed in the nucleus.)  Explain why this method would copy the mRNA but not the rRNA, tRNA,  or other RNA molecules present.

2. What would be the effect of contaminating DNA in our RNA preparations upon the cDNA synthesis?

3. Would this method, using oligo dT primed cDNA synthesis, be suitable for use with prokaryotic total RNA preparations?

3. In the formation of the cDNA, we will be incorporating molecules of fluorescent dyes called Cy3 and Cy5.  Cy3 fluoresces green and Cy5 fluoresces red (that’s not the colors they look like, but the colors of fluorescent light they emit when excited).  Each of these dyes will be added to the reaction mixture coupled to dUTP. 
   1. What base will each of these dye-coupled nucleotides pair with?

2. Since the bulky dye slows the cDNA synthesis, what do you think might  be done to incorporate dye but still keep the speed of synthesis up? 

3. Diagram how you would set up the experiment so that the Cy3 dye will be attached to the cDNA from the glucose/0.7 cells and the Cy5 dye attached to the cDNA from the ethanol/6.0 cells?

4. If you started with the SAME RNA for the two fluorescently labeled preparations, mixed together the two cDNA preparations, and hybridized them with the same microarray slide containing all the yeast genes, can you think of any reasons why they should not hybridize to each spot with exactly the same green and red fluorescence intensities (ratio of 1.000000)?  (No fair answering ‘experimental error”; be specific!)

4. Read the 1997 paper by Joe DeRisi (pdf sent along with this document and/or available from Stanford Microarray Database) on gene expression changes in yeast diauxie.  The diauxie means ‘two foods’ and refers to the use of glucose first, producing ethanol by non-oxygen requiring pathways, and then the aerobic utilization of ethanol.  There is no need to change the medium; it naturally happens over time.  We will be using total RNA prepared from yeast early in the logarithmic growth period (A600 = 0.7) and total RNA prepared from yeast late in growth near stationary phase (A600 = 6.0).  

a. After you have read the DeRisi paper, using the last set of graphs in the paper

and, if you wish the Saccharomyces Genome Database, at the web site:

http://www.yeastgenome.org/ write down three genes you predict would be high

and three genes you would predict would be low in expression when using

glucose (0.7) and when using ethanol aerobically (6.0).  Use the three letter gene

name and the yeast gene identifier for each one.  (Example: ENO1, YGR254W) 

0.7 A600; glucose use                        6.0 A600; aerobic ethanol use

predicted high expression:                     predicted high expression:

predicted low expression:                      predicted low expression:

b.      For each of these genes given above, describe the molecular function and the biological process in which the gene is involved.  You may use the SGD shorthand versions or read the longer descriptive paragraphs and give a longer summary.

5. In microarray data, genes from the same pathway often are co-regulated.  What kinds of mechanisms could result in coordinate transcriptional control of all the genes in the same pathway?  

6. In microarray data, duplicate samples do not always provide the same green/red ratios in the output data. 
   1. What are some of the reasons why they might not?  How could these reasons be best addressed in figuring out the meaning of apparent changes in gene expression?

2. What controls can be used to address these problems?

3. How do these problems confound the interpretation of apparent changes in gene expression?

7. The microarray expression technique we will be using generates data in the form of ratios of mRNA signals from cells grown under two conditions.  People talk informally about the results as if they show that mRNA is induced under some condition and repressed under some other condition.  However, this method does not measure the absolute amount of mRNA present, nor does it measure how fast it is being synthesized or broken down.  Rather, it measures the ratio of the ‘standing crop’ of the total mRNA present under two different conditions. 
   1. Explain what we are actually measuring with this method?  Explain why this insight makes the choice of a control sample absolutely critical to the interpretation of the results.

2. What factors besides mRNA concentration affect the level of the functional protein product of a gene?

8. The investigators at Institute for Systems Biology in Seattle have found that only around 60% of the changes they see in mRNA hybridization on microarray chips correspond to changes in the cellular concentration of the same protein encoded by the mRNA.  Given what is discussed in question 6 and question 7, why do you think people are still using this technique; i.e. what can it contribute to our understanding compared with other methods for examining regulatory events?