A simple semisolid subtraction method using carbodiimide-coated microplates.

Abstract

In this article, we develop a novel subtraction method using carbodiimide-bound microplates. This method utilizes the high affinity of carbodiimides for both single- and double-stranded nucleic acids. Carbodiimide-mediated end-attachment of driver RNA to microplates allows semisolid phase hybridization between driver RNA and target cDNA, and ensures easy removal of RNA/cDNA hybrids composed of the genes commonly expressed in driver and target. As a result, the target-specific genes are left unhybridized and enriched in the hybridization supernatant. We define the optimal conditions for the method as a target/driver RNA ratio of 1:10 and a period of hybridization of 24 h. There are at least three major advantages with the present method: (1) The entire procedure, which consists of two steps, is very simple; (2) hybridization efficiency can be monitored before further processing of the samples; and (3) rare transcripts can be effectively enriched. This method may be a powerful tool to isolate the genes specifically expressed in particular cell or tissue types, and is easily applicable to many studies in molecular biology and genetics. Isolation of polyploid megakaryocyte-specific genes is shown as an example.