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of protein activity was not required (
Wang & Brown, 1991
). These screens
found between 14 and 34 genes in tail, hind limb, intestine, and brain
(
Buckbinder & Brown, 1992; Denver, Pavgi, & Shi, 1997; Shi & Brown,
1993; Wang & Brown, 1993
). Massive sequencing efforts on ESTs, cDNA
libraries, and genomic DNA in frogs led to increasing numbers of genes
known, ultimately making virtually all genes known. Microarray technol-
ogy allows the expression of many or all genes to be examined simulta-
neously in a single experiment (
Fig. 12.1
). The first microarray for frog
Array platforms
cDNA/PCR product array
Agilent oligoarray
Affymetrix GeneChip
Probe source
PCR products
Oligosynthesis
Oligosynthesis
200-600 base pairs
Custom-built, spotted on
membrane or slide
60 base pairs
25 base pairs
Probe length
Chip
production
Commercial,
in situ
synthesis on slide
Commercial,
in situ
synthesis on slide
~14,400,
X. laevis
~29,900,
X. laevis
2.0
~51,000,
X. tropicalis
No. of
transcripts
~400-1000 selected
~21,500,
X. laevis
Reference sample experimental design
Label with
cy5
vs.
cy3
Mix and
hybridize
Total RNA
Figure 12.1 Types of microarray platforms and example microarray experimental
design. Top panel indicates characteristics of the microarray platforms used in studies
of frog metamorphosis. Lower panel depicts an experimental design using a reference
sample, commonly used with the Agilent platform. The reference sample is a single
preparation of RNA from a mixture of tissues and stages and labeled with one color
(e.g., the red fluorescent dye, cy5). The experimental samples, including treatment
and control samples, are labeled with a different color (e.g., the green fluorescent
dye, cy3). Each experimental sample is mixed with a portion of the reference sample
and hybridized to the microarray slide, resulting in each spot on the array with levels
of red and green fluorescence corresponding to the level of expression of the RNA
in the reference versus experimental sample. Expression differences among experimen-
tal samples are determined based on expression levels relative to the reference sample.
The other platforms commonly use a one-color design and compare intensity values
directly among samples.
