Cross-species hybridization: characterization of gene expression in woodchuck liver using human membrane arrays.
Total RNA from normal adult woodchucks was analyzed using membrane arrays containing human cDNA clones, and the gene expression patterns were compared to human liver. Various hybridization and wash conditions were examined. In both the woodchuck and human livers, 352 genes were identified as highly expressed (Z-scores > or =1.96). These genes represented numerous liver functions: transcription, RNA processing, signal transduction, protein synthesis and degradation, as well as enzymes. Several genes were selected and expression was verified by Northern blots for woodchuck liver. There were no false positives but 29 genes were identified as false negatives, expressed only in human liver. Possible reasons for these false negatives were the length and percentage of homology between the two species, differences in the distribution and types of mismatches, and the sequence region spotted on the array. These were assessed by examining expression of the transferrin gene in both species. A 200-fold range of RNA concentration (0.1-20 microg total RNA) was also examined and the optimal RNA concentration was determined to be 5 microg. Membranes were capable of being hybridized and reprobed at least five times. The study demonstrates that cross-species hybridization is a valid method for identifying gene expression in woodchuck liver.
Division of Molecular Virology and Immunology, Department of Microbiology and Immunology, Georgetown University Medical Center, Rockville, Maryland 20850, USA. rinaudja@georgetown.edu
Jo Ann Spiewak
JA
Rinaudo
John L
JL
Gerin
D000818:Animals; D001483:Base Sequence; D020869:Gene Expression Profiling; D006801:Humans; D006824:Hybridization, Genetic; D008099:Liver / Q000378:metabolism*; D008392:Marmota / Q000378:metabolism*; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D013045:Species Specificity; D014168:Transferrin / Q000737:chemistry / Q000235:genetics
D011506:Proteins; D014168:Transferrin