Target Discovery and Validation Reviews and Protocols

118 Røsok and Sioud
Compared with subtraction cloning, positive selection can be more efficient for
isolating differentially expressed transcripts because subtractive hybridization
rarely goes to completion. Therefore, a background of common unsubtracted
clones may persist. In contrast, because tester concentration is low, the desired
tester–tester duplexes take longer to form than driver–driver duplexes. Thus, rare
clones that have not reannealed are likely to be missed in positive selection. A
solution to this problem is to use reagents, which decreases the aqueous volume,
and thus increases the hybridization rate. Polyethylene glycol and phenol may
serve this purpose (13). Using a normalized tester population also makes positive
selection more effective. The positive selection may be done without including a
PCR step. More recent methods include PCR, which is done in representational
difference analysis (RDA) and in subtractive suppression hybridization (SSH).
1.1.5. Representational Difference Analysis (RDA)
In RDA, target cDNA fragments from one of two cell populations are
sequentially enriched by favorable hybridization kinetics and subsequently
amplified by PCR, whereas material common to both populations is eliminated
by selective degradation (14).
RDA, originally developed as a method for isolation of differences between
two complex genomes (15), has further been developed to cDNA RDA. This technique
is a PCR-based subtractive hybridization technique for identifying differentially
expressed genes. The mRNA from both populations is isolated and converted
into dscDNA. By cutting this cDNA with a four-base pair (bp) restriction enzyme,
a large proportion of the transcripts will be represented as amplifiable fragments
(150 bp–1.2 kilobases [kb]) (16). Amplified fragments, known as “representations,”
are hybridized at high driver:tester ratio. By using a combination of
linker ligation, restriction cutting, and PCR, exponential amplification of only
tester–tester hybrids is achieved. Single-stranded DNA is enzymatically degraded.
To amplify only true differences, a second and often a third round of subtraction
is performed, in which the ratio of driver to tester is increased. Difference
products are visible in agarose gels and can easily be subcloned. Transcripts
expressed in less than one copy per cell can be detected with low rate of false
positives (14). In circumstances where many differences are expected, RDA
enriches the products that amplify most effectively, and not necessarily all the
interesting differences. Thus, RDA is not the method of choice when investigating
tissues with abundant differences (14). Because no normalization step is
included, the method is not efficient for the detection of rare transcripts.
1.1.6. Suppression Subtractive Hybridization (SSH)
In SSH, normalization and subtraction are combined in a single step to
equalize the abundance of cDNA within the target population and to exclude the