Subtractive hybridization strategy using paramagnetic oligo(dT) beads and PCR.
Subtractive hybridization has been widely used for the identification of differentially expressed genes. Here we describe a simple, sensitive strategy of subtractive hybridization that involves binding the driver poly(A)+ RNA pool to paramagnetic Dynabeads Oligo (dT)25. After hybridization with target cDNA, the molecules common to both pools are removed. The subtracted cDNA is then amplified with PCR and used for library screening. Using this method, we have identified four cDNA clones that represent developmentally regulated transcripts in the central nervous system of the tobacco hornworm Manduca sexta. All four transcripts are of low abundance, comprising only 0.001%-0.5% of the poly(A)+ RNA pool.
Mészáros
M
M
University of Arizona, Tucson 85721, USA.
Morton
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D000818:Animals; D015152:Blotting, Northern; D003001:Cloning, Molecular / Q000379:methods; D018076:DNA, Complementary / Q000302:isolation & purification*; D018507:Gene Expression Regulation, Developmental; D015723:Gene Library; D007814:Larva; D008280:Magnetics; D018402:Manduca / Q000235:genetics / Q000254:growth & development; D008675:Metamorphosis, Biological / Q000235:genetics; D008863:Microspheres; D009420:Nervous System / Q000378:metabolism; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011679:Pupa; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D013382:Subtraction Technique
D018076:DNA, Complementary; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; C027903:oligo (dT)