Target Discovery and Validation Reviews and Protocols

Pancreatic Cancer 59
on the microarray, the ratio of fluorescence intensities reflects the relative
abundance for that mRNA between the two samples.
The two-color fluorescence format provides robust measurements of gene
expression ratios, effectively mollifying variations in amounts of spotted DNA
and of hybridization kinetics. In some experimental designs, the selection of the
second-color “reference” specimen is natural, for example, the zero time-point
in a time-course, or the untreated sample in a pharmacological treatment. In
other cases, an arbitrary “universal” RNA reference, comprising a pool of mRNA
from cell lines or tissues, is more appropriate (28). Because each sample is
hybridized against the same reference, samples can be readily compared with
each other. In such cases, gene expression ratios are typically “mean-centered,”
i.e., reported in relation to the average of the specimens assayed (rather than to
the arbitrary reference).
For small specimens (e.g., tissue biopsies or microdissected samples),
insufficient input RNA may be available for the labeling protocol detailed
in Subheading 3.1.3. In such cases, the reader is directed to linear RNA
amplification protocols based on T7 RNA polymerase-mediated in vitro
transcription (29).
1.3. Serial Analysis of Gene Expression
SAGE is a comprehensive expression profiling technology for quantitative
gene expression in samples that does not depend on the prior availability of
transcript information (24). In this aspect, it varies from other expression
profiling technologies such as cDNA microarrays (see Subheading 2.1.2. and
Chapter 5, Volume 1) where analyses is limited to a known repertoire of arrayed
sequences. The premise of SAGE rests on the concept that a short sequence of
nucleotides (~11 base pairs [bp], known as a “tag”) is sufficient for uniquely
identifying a transcript (24,30). The “serial” aspect of this platform comes
from the generation of “concatemers” by ligating large numbers of SAGE tags
within a “library” that are then serially analyzed by sequencing for gauging relative
transcript levels.
The sequential steps involved in generation of a SAGE library are illustrated
in Fig. 1 (30). Briefly, double-stranded cDNA is generated from mRNA
isolated from a sample of interest and immobilized on magnetic beads. The
cDNA is then digested with a frequent cutting restriction enzyme such as
NlaIII and ligated to a linker with a restriction recognition site and a PCR
primer site. Subsequent digestion by another restriction enzyme (e.g., BsmfI)
generates tags that contain the linker and a short nucleotide sequence (~11 bp)
downstream of BsmfI recognition site that is specific for the transcript from
which it was generated. Pairs of tags are then ligated to generate so-called
“ditags,” which are PCR amplified, digested by NlaIII to remove the linkers