Target Discovery and Validation Reviews and Protocols

126 Røsok and Sioud
splicing may be detected effectively as well (58). Problems concerning limiting
number of known transcripts will in a short time be negligible for important
model organisms because genome projects for several species are completed.
Identification of all transcribed genes of model organisms initiates the production
of arrays harboring every gene for the specific organism and thereby
increases the potential of the method.
1.3. Rescue–Suppression-Subtractive Hybridization
As discussed in Subheading 1, several techniques have been developed to
identify the differences in gene expression between samples from different
biological materials. Suppression-subtractive hybridization is one of the techniques
that enables the enrichment of nucleic acids representing genes that are
uniquely or abundantly expressed in cells of interest (7). And as explained
above (Subheading 1.1., item 2), due to the suppression PCR effect, only
dscDNA molecules with two different adaptors can be exponentially
expressed; thus, transcripts differentially expressed in one cell population are
enriched. Despite the identification of differentially expressed genes by using
SSH, a high number of false positives are reported (30). False positives are
transcripts that seem to be differentially expressed by using one technique, but
that are unable to be confirmed by using other independent methods, e.g.,
Northern, the gold standard for verification of differential expression.
Sometimes, these transcripts may represent as much as 50–90% of the identified
candidates (31), especially when two closely related RNA populations are
being compared. Thus, the most time-consuming step is the confirmation that
the isolated cDNA clones represent differentially expressed genes. By combining
PCR-based cDNA subtraction and Northern blotting, truly differentially
genes can be quickly identified (Fig. 1). Notably, this novel strategy enables
integration of the time-consuming confirmation of candidates for differential
expressed transcripts in the selection stage (21).
References
1. Saiki, R. K., Scharf, S., Faloona, F., et al. (1985) Enzymatic amplification of betaglobin
genomic sequences and restriction site analysis for diagnosis of sickle cell
anemia. Science 230, 1350–1354.
2. Kacharmina, J. E., Crino, P. B., and Eberwine, J. (1999) Preparation of cDNA
from single cells and subcellular regions. Methods Enzymol. 303, 3–18.
3. Liu, M., Subramanyam, Y. V., and Baskaran, N. (1999) Preparation and analysis
of cDNA from a small number of hematopoietic cells. Methods Enzymol. 303,
45–55.
4. Sambrook, J., Fritsch, E., and Maniatis, T. (1989) Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring, NY.