Low-ratio hybridization subtraction.
A hybridization subtraction protocol that uses low ratios of RNA to cDNA has been developed to enrich for the cDNA of transcripts that are elevated in one cell population relative to another. This low-ratio hybridization subtraction protocol was found to yield substantial enrichment for the cDNA of low-abundance transcripts induced or increased only several fold. Conditions for the cloning of cDNA enriched by our hybridization subtraction and identification of clones coding for induced transcripts are presented. By screening the cDNA library with probes synthesized from the starting cDNA and cDNA enriched by low-ratio hybridization subtraction, clones coding for induced transcripts could be efficiently identified. The choice of reverse transcriptase used to synthesize the cDNA was found to be important for the enrichment of cDNA for longer length RNA. Low-ratio hybridization subtraction of cDNA synthesized with MMLV reverse transcriptase was effective for the enrichment of cDNA coding for RNA to at least 5 kb in length, while the AMV enzyme was effective only for the cDNA of shorter RNA (less than 1 kb). The characterization of several different low-ratio hybridization subtraction libraries is presented, and the advantages and disadvantages of various hybridization subtraction strategies are discussed.
Laboratory of Molecular Genetics, NIA, NIH, Baltimore, Maryland 21224.
J
J
Fargnoli
N J
NJ
Holbrook
A J
AJ
Fornace
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000096:biosynthesis*; D015698:Genomic Library; D008297:Male; D009693:Nucleic Acid Hybridization; D012333:RNA, Messenger / Q000096:biosynthesis; D012194:RNA-Directed DNA Polymerase; D051381:Rats; D011919:Rats, Inbred Strains
D012333:RNA, Messenger; D004247:DNA; D012194:RNA-Directed DNA Polymerase