Identification of differentially expressed genes by mutually subtracted RNA fingerprinting.
A mutually subtracted RNA fingerprinting (SuRF) method has been developed that allows efficient identification of differentially expressed sequence tags between two samples. Mutual subtractions of two RNA samples are achieved by first synthesizing cDNAs using oligo(dT) coupled with magnetic beads which are then reciprocally hybridized to starting RNA samples to remove common 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 gene expression pertinent to osteogenic sarcoma which was achieved by employing the method between FOB (an immortalized fetal osteoblast) and MG63 (an osteosarcoma) cell lines. An estimated 10% of the total expressed genes in these two cell types were screened by the method. This analysis identified 96 differentially expressed sequences, none of which was identified repeatedly. A subset of these sequences was subsequently confirmed to have differential 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.
Fuchs
B
B
Department of Orthopedics, Mayo Clinic and Foundation, Rochester, Minnesota 55905, USA.
Zhang
K
K
Bolander
M E
ME
Sarkar
G
G
D018076:DNA, Complementary / Q000378:metabolism; D004272:DNA, Mitochondrial / Q000378:metabolism; D020224:Expressed Sequence Tags; D005821:Genetic Techniques; D006801:Humans; D010006:Osteoblasts / Q000378:metabolism; D012516:Osteosarcoma / Q000235:genetics; D008249:Protein-Lysine 6-Oxidase / Q000096:biosynthesis / Q000235:genetics; D012313:RNA / Q000032:analysis* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D004272:DNA, Mitochondrial; D012333:RNA, Messenger; D012313:RNA; D008249:Protein-Lysine 6-Oxidase