A simple semisolid subtraction method using carbodiimide-coated microplates.
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.
Kikuchi
J
J
Division of Molecular Hematopoiesis, Jichi Medical School, Tochigi, Japan.
Furukawa
Y
Y
Suzuki
O
O
Hayashi
N
N
Nakamura
M
M
Morita
M
M
Matsuda
M
M
D015152:Blotting, Northern; D002234:Carbodiimides / Q000378:metabolism*; D020170:Caspase 1 / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007375:Interleukin-1 / Q000235:genetics / Q000378:metabolism; D007948:Leukemia, Monocytic, Acute; D009693:Nucleic Acid Hybridization / Q000379:methods*; D012333:RNA, Messenger / Q000302:isolation & purification / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D002234:Carbodiimides; D018076:DNA, Complementary; D007375:Interleukin-1; D012333:RNA, Messenger; D020170:Caspase 1; D013755:Tetradecanoylphorbol Acetate