Target Discovery and Validation Reviews and Protocols

36 Imoto et al.
2. Materials
Two types of cDNA microarray measurements of S. cerevisiae have been
prepared for our purpose. One type is the microarray data obtained by gene disruptions,
and the other type is time-course data of responses to an antifungal
medicine. The number of disruptants is 120 for the first type of data, and the
time-course data for one dose consists of five microarrays.
2.1. Microarray Data of Single Gene Disruptions
The first microarray data are obtained by disrupting 120 genes, where
mostly transcription factors are disrupted. The BY4741 (MATa, HIS3D1,
LEU2D0, MET15D0, URA3D0) is used as the wild-type strain, and purchased
gene disruptions were from Research Genetics (Huntsville, AL). The disrupted
genes are listed in Table 1. To monitor the gene expression profile, cells were
inoculated and grown in YPD medium (1% yeast extract, 1% bacto-peptone,
2% glucose) at 30ºC until optical density600 reached 1.0 in the logarithmic
growth phase, and then cells were harvested to isolate mRNA for assay of gene
expression. The respective parental strain was the control used for each disruptant
strain. See Aburatani et al. (14) for details. The total RNAs are extracted
from these experiments and labeled by Cy5. These Cy5-labeled RNAs are
hybridized with Cy3-labeled RNAs from nontreated cells. A microarray then
measures 5871 gene expression levels at once.
2.2. Drug Response Time-Course Microarray Data
To determine the previously unknown underlying molecular affects of the
popular generic antifungal agent griseofulvin, we created time-course drug
response microarray data. Griseofulvin is a widely prescribed oral antifungal
agent that is indicated primarily for severe fungal infections of the hair and
nails. Although griseofulvin’s molecular action is unknown, it is known that the
drug disrupts mitotic structure in fungi, leading to metaphase arrest.
We incubated yeast cultures in dosages of 10, 25, 50, and 100 mg/mL antifungal
medication in culture and took aliquots of the culture at five time-points
(0, 15, 30, 45, and 60 min) after addition of the agent. Here, time 0 means the
start point of this observation and just after exposure to the drug. We then
extracted the total RNAs from these experiments, labeled the RNAs with Cy5,
hybridized them with Cy3-labeled RNAs from nontreated cells, and applied
them to full genome cDNA microarrays, thereby creating a data set of 20
microarrays for drug response data.
2.3. Data Normalization
It is known that expression profile data measured by microarrays have several
systematic biases, such as spotting position-specific bias, intensity dependent