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
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).
- Please answer the following with regard to your plans
to use microarrays in an undergraduate class.
- How will microarray use relate to the overall
goals of your class?
- What concerns do you have about using this type of
experiment in your class?
- If you have chosen a type of microarray experiment
that you would like your class to perform, what kind?
- How much time are you planning to devote to
microarray experiments? To microarray data analysis?
- What contingencies do you have in mind for
problems that may arise with your microarray experiments?
- 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
- 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.
- What would be the effect of contaminating DNA in
our RNA preparations upon the cDNA synthesis?
- Would this method, using oligo dT primed cDNA
synthesis, be suitable for use with prokaryotic total RNA preparations?
- 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.
- What base will each of these dye-coupled
nucleotides pair with?
- 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?
- 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?
- 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!)
- 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
genes you would predict would be low in expression when using
(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
expression: predicted high expression:
expression: predicted low expression:
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
- 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?
- In microarray data, duplicate samples do not always
provide the same green/red ratios in the output data.
- 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?
- What controls can be used to address these
- How do these problems confound the interpretation
of apparent changes in gene expression?
- 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.
- 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.
- What factors besides mRNA concentration affect the
level of the functional protein product of a gene?
- 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?