Suppression subtractive hybridization technology

This chapter describes suppression subtractive hybridization (SSH)—a sophisticated cDNA subtraction method to enrich and isolate differentially expressed genes. SSH accomplishes normalization and subtraction by taking advantage of the different rates of hybridization of cDNA strands for different genes depending on their abundance level and the degree of (differential) expression. SSH may be used for pairwise treatment comparisons and must be replicated with the tester and driver reversed to identify gene expression changes in both directions. It is not a quantitative method for measuring expression differences. SSH is best used for identifying genes that are completely absent, rather than expressed less abundantly, in the driver sample. One of the major problems associated with specific cellular characterization is the low amount of sample. However, problems associated with tissues in small quantities can be solved by a restricted polymerase chain reaction (PCR) amplification step prior to cDNA subtraction. When the amount of starting material is limited, it is possible to start with only a few nanograms of total RNA and produce enough double-stranded cDNA of both tester and driver to subtract two specific cell populations by using PCR technology.