Methods

Whether to produce subtracted cDNA libraries or screen standard libraries with subtracted probes is the first consideration one must address. We advocate the construction of representative cDNA libraries that can later be screened with any desired probe. Indeed, such libraries can also be used to produce either synthetic driver or target mRNAs in large quantitites. This approach has the advantage that only one or two libraries must be constructed for each target/driver pair, and any type of probe can be used as may later be required. It has the disadvantage that more clones must be screened to ensure the representation of the rarest mRNAs.

The choice of subtraction protocol to be followed depends on the availability of mRNA from both target and driver cells or tissues. If the driver mRNA is not limiting (~50 ^g available) then one can begin with Subheading 3.7., photobiotinylation of driver mRNA, otherwise one should first construct a library which may then be used to generate driver RNA. Similarly, the abundance of target mRNA and the desire to produce a representative or subtracted library will dictate whether the target will be oligodT-primed first strand cDNA or random primed cDNA produced after in vitro transcription of the library (Fig. 1).

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