Detection of ribonuclease H-generated mRNA fragments in human leukemia cells following reversible membrane permeabilization in the presence of antisense oligodeoxynucleotides.
The involvement of ribonuclease H (RNase H) in antisense phenomena in intact cells has, to date, only been adequately demonstrated for microinjected Xenopus systems. The significance of RNase H for the antisense effects of oligodeoxynucleotides observed in human and other mammalian cell cultures has remained obscure, in part because of inadequate analytic methods. In this report we show that the "reverse ligation-mediated PCR" (RL-PCR) procedure permits amplification of RNA fragments produced by oligodeoxynucleotide-directed RNase H activity. We have used this procedure to demonstrate RNase H-dependent antisense effects in irreversibly permeabilized (dead) cells and reversibly permeabilized (live) cells.
Giles
R V
RV
Department of Biochemistry, University of Liverpool, UK.
Spiller
D G
DG
Tidd
D M
DM
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002463:Cell Membrane Permeability; D017931:DNA Primers; D006801:Humans; D007951:Leukemia, Myeloid; D008845:Microinjections; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000008:administration & dosage / Q000494:pharmacology*; D009843:Oligoribonucleotides / Q000032:analysis*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014981:Xenopus
D017931:DNA Primers; D016376:Oligonucleotides, Antisense; D009843:Oligoribonucleotides; D012333:RNA, Messenger; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H