Paul B. Fisher

5 - Identification of Differentially Expressed Genes Using Rapid Subtraction Hybridization: Detailed Methodology for Performing Rapid Subtraction Hybridization

This chapter presents a protocol for subtracted complementary DNA (cDNA) library construction by rapid subtraction hybridization (RaSH). Additional methods, including reverse Northern and Northern Blot hybridization, which are part of the RaSH approach, are described in the chapter. Proof-of-principle for the RaSH approach was obtained by analyzing the temporal changes of gene expression occurring during the induction of terminal differentiation in human melanoma cells treated with interferon-beta plus mezerein. In RaSH, complementary DNA (cDNA) samples are restriction-digested with frequent cutters, such as DpnII , to an average size of 256 bp for improving hybridization efficiency. Moreover, the restriction digestion of tester cDNA only prior to hybridization enables the selection and cloning of tester-unique cDNAs by a direct ligation of hybridization mixtures to corresponding sites in the plasmid vector. Thus, RaSH reduces the amount of starting materials required for conventional subtraction hybridization by one tenth by using polymerase chain reaction (PCR) and obviates the need to separate and clone subtracted cDNA species into plasmid vector or bacteriophage, a nontrivial, technically demanding step in subtractive cDNA library construction.