Direct labeling of RNA with multiple biotins allows sensitive expression profiling of acute leukemia class predictor...

Direct labeling of RNA is an expedient method for labeling large quantities (e.g. micrograms) of target RNA for microarray analysis. We have developed an efficient labeling system that uses T4 RNA ligase to attach a 3'-biotinylated donor molecule to target RNA. Microarray analyses indicate that directly labeled RNA is uniformly labeled, has higher signal intensity than comparable labeling methods and achieves high transcript detection sensitivity. The labeled donor molecule we have developed allows the attachment of multiple biotins, which increases target signal intensity up to 30%. We have used this direct-labeling method to detect previously discovered class predictor genes for two types of cancer: acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). In order to test the sensitivity of direct RNA labeling, we analyzed the AML and ALL expression profiles for predictor genes that were previously found to show elevated expression in the disease state. Direct labeling of AML poly(A) RNA detects 90% of the class predictor genes that are detected by the IVT-based target amplification method used to discover the genes. These results indicate that the detection sensitivity, simplicity (single tube reaction) and speed (2 h) of this direct labeling protocol may be ideal for diagnostic applications that do not require target amplification.