Cloning Differentially Expressed Genes by Using Differential Display and Subtractive Hybridization
Differential gene expression occurs in all phases of life, including development, maintenance, injury, and death of an organism. Being able to identify these genes will help understand not only gene function but also the underlying molecular mechanisms of a particular biological system. Differential display (DD) (1,2) and subtractive hybridization (SH) (review see refs. 3, 4, 5) are by far the most common methods currently used by investigators for this purpose. However, do these techniques identify both abundant and rare mRNAs? Previous kinetic studies (6,7) indicate that approx 98% of all mRNA species within the cell have a prevalence ranging from 1/10,000 to 1/100,000, and thus are considered rare. Furthermore, the remaining approx 2% of mRNA species (about 200–500) contribute to over 50% of the total mRNA mass (6,7). Therefore, it is relatively easy to identify a few very abundant differentially expressed mRNAs (i.e., using plus/minus screening); but if the quest is to identify the majority of differentially expressed mRNA species, then a method that is not sensitive to mRNA abundance is required. To evaluate how well DD and SH can identify abundant as well as rare differentially expressed mRNAs, we used both of these methods to find differentially expressed mRNAs within HeLa cells in response to interferon-γ. Described below is a summary of our results (see ref. 8 for original report), followed by detailed protocols of DD, SH, and reverse Northern.
KeywordsVortex Manifold EDTA MgCl2 Gelatin
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