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222 Bartlett
Notwithstanding these caveats, SAGE analysis is a highly powerful experimental
tool on which is increasingly applied to transcriptome analysis. The power of SAGE
analysis is the product of an extremely complex experimental protocol, one that relies
on the construction of a representative cDNA library for each RNA sample to be
analyzed. In addition, the number of clones that must be sequenced for even a simple
comparison between two libraries, despite the use of short sequence tags, is high
(between 800–4000 has been suggested). Furthermore, although there is a high
probability that a short sequence tag of 10 bp will identify a unique sequence, it is
undeniably more complex to identify gene transcripts from such tags. Proponents
of SAGE point out, with some justification, that the effort may well repay the cost.
Nonetheless, the prospect of performing even a limited analysis of clinical material
(50–100 tumors) using this method is a daunting one and many will be tempted to take
a simpler, albeit less quantitative approach, such as microarray or differential display
analysis, for their first steps in transcriptome analysis. More importantly, SAGE is
an undirected technique that produces a global transcriptome map. Both microarrays
and differential display techniques can, however, be readily tailored to the particular
experimental question and hypothesis under investigation. As discussed at the outset of
this chapter, careful consideration will be required to select the method most appropriate
to the research question, expertise, and resources available to the investigator.
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