Whole Cells - Molecular Theory of the Living Cell - page 383

that are deep and powerful enough to enable cell biologists to correctly
analyze and interpret the avalanche of DNA array data that is accumulating on
the Internet.

12.2 The DNA Microarray Technique

cDNA fragments can be fabricated into either microarrays or macroarrays
(Garcia-Martinez et al. 2004), but the term “microarrays” frequently refer to both
micro- and macroarrays . A microarray consists of a microscopic slide (or its
equivalent), about 2

2 cm in dimension, divided into, typically, 10,000 squares,
each of which covalently binds hundreds of copies of a fragment of DNA (i.e.,
cDNA, or oligonucleotides) that is complementary to a stretch of the genome
encoding a RNA molecule (Watson and Akil 1999). Thus, using one microarray,
it is possible to measure simultaneously the levels of 10,000 RNA molecules in a
biological sample. Before the development of the microarray technique, it was
possible to study only a small number of RNA molecules at a time. The experimental
procedures involved in DNA microarray measurements are (Watson and Akil 1999):

1. Isolate RNA from broken cells.
2. Synthesize fluorescently or radioactively labeled cDNA from RNA using reverse
transcriptase and fluorescently or radioactively labeled nucleotides. When fluo-
rescently labeled nucleotides are employed, the red fluorophore Cy5 is used to
label the DNA synthesized from the RNA isolated from query samples and the
green fluorophore Cy3 is used to label the cDNA synthesized from the RNA
isolated from reference samples.
3. Prepare a microarray either with EST (expressed sequence tag, i.e., DNA
sequences, several hundred nucleotides long that are complementary to the
stretches of the genome encoding RNAs) or oligonucleotides (synthesized
right on the microarray surface).
4. Pour the labeled cDNA preparation over the microarray surface to effect
hybridization and wash off excess debris.
5. Measure the light intensity or the radioactivity of the labeled cDNA bound to
individual squares (or spots) on a microarray surface using a computer-assisted
microscope.
6. Display the final result as a table of numbers, each registering the signal intensity
of a square on the microarray which is proportional to the concentration of
cDNA (and ultimately RNAs in the cell before breaking the cell membrane)
located at row x and column y. Row x indicates the identity of the genes
encoding RNAs being measured, and column y indicates the time of measure-
ment or the conditions under which the RNA levels are measured.

Typical results of microarray measurements of RNA levels are shown in Figs. 9.1 ,
12.1 ,and 12.2 . These data were measured by Garcia-Martinez et al. (2004) from
budding yeast Saccharomyces cerevisiae undergoing glucose-galactose shift at