Subtractive Hybridization for the Isolation of Differentially Expressed Genes Using Magnetic Beads

Subtractive hybridization methods provide a means to isolate genes that are specifically expressed in a cell type or tissue (1 –3 ), genes that are differentially regulated during activation or differentiation of cells (4 –7 ), or genes that are involved in pathological conditions such as cancer (8 –10 ). Such methods are suitable for the isolation of low-expression genes. The principle of these approaches is to remove the mRNA species common to different cell types or tissues by subtraction, leaving the cell type/tissue-specific mRNAs for further manipulation and analysis. Alternatively, mRNA from both the subtractor and target population can be reverse transcribed and amplified by PCR followed by subtractive hybridization and isolation of specific sequences (9 ). In the last 20 yr, several subtractive strategies have been described (2 –5 ,9 ), but subtraction hybridization/cloning still remains a technically demanding, time-consuming, and labor-intensive technology, including the need for large amounts of mRNA or highly purified single-stranded DNA. Several subtractive hybridization strategies based on solid-phase hybridization on magnetic Dynabeads have previously been described (11 –14 ). In this chapter, we present one such solid-phase strategy developed by us (12 ) that is an improved version of an already published method (12 ). Our approach takes advantage of the properties of magnetic Dynabeads allowing simple and rapid buffer changes required for optimal hybridization and enzymatic reactions.