Identification of differentially expressed genes by mutually subtracted RNA fingerprinting

A mutually subtracted RNA fingerprinting (SuRF) method has been developed t hat allows efficient identification of differentially expressed sequence ta gs between two samples. Mutual subtractions of two RNA samples are achieved by first synthesizing cDNAs using oligo(dT) coupled with magnetic beads wh ich are then reciprocally hybridized to starting RNA samples to remove comm on mRNAs between them. The second step involves differential fingerprinting of the subtracted RNA samples by polymerase chain reaction with specially designed degenerate primers, SuRF was applied to identify alteration in gen e expression pertinent to osteogenic sarcoma which was achieved by employin g the method between FOE (an immortalized fetal osteoblast) and MG63 (an os teosarcoma) cell lines. An estimated 10% of the total expressed genes in th ese two cell types were screened by the method. This analysis identified 96 differentially expressed sequences, none of which was identified repeatedl y. A subset of these sequences was subsequently confirmed to have different ial expression between the two cell types. Removal of common mRNAs prior to differential display should diminish redundant identification of abundant genes and increase the chance of identifying rare differentially expressed genes. (C) 2000 Academic Press.