In situ reverse transcription for detection of hybridization between oligonucleotides and their intracellular targets.
It is often important to know that a phenotypic change caused by antisense treatment has occurred because the antisense molecule has specifically hybridized to its intracellular target, rather than by some nonspecific, indirect route. We describe here a method that can be used to detect hybridization of an antisense oligodeoxynucleotide to its intracellular target RNA and, furthermore, to identify the sites at which hybrids are located in situ. Oligodeoxynucleotides are first taken up by the live cell and then cells are fixed and subjected to an in situ reverse transcription reaction. The reverse transcription assay exploits the fact that only oligonucleotides that are hybridized to RNA will act as primers for reverse transcriptase and allow incorporation of labeled nucleotide into cDNA; unhybridized oligonucleotides will not prime reverse transcription. We illustrate this approach by comparing the levels of oligo(dT) hybridized to poly(A) RNA in cells that have taken up the oligo(dT) with and without cationic lipid in the medium.
Politz
J C
JC
Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA. Joan.Politz@umassmed.edu
Singer
R H
RH
D000818:Animals; D002460:Cell Line; D005456:Fluorescent Dyes; D017404:In Situ Hybridization, Fluorescence; D008081:Liposomes / Q000378:metabolism; D009693:Nucleic Acid Hybridization / Q000379:methods*; D009838:Oligodeoxyribonucleotides / Q000032:analysis / Q000378:metabolism; D020319:Oligodeoxyribonucleotides, Antisense / Q000235:genetics*; D011067:Poly dA-dT / Q000032:analysis; D012313:RNA / Q000235:genetics; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D051381:Rats; D014158:Transcription, Genetic
D005456:Fluorescent Dyes; D008081:Liposomes; D009838:Oligodeoxyribonucleotides; D020319:Oligodeoxyribonucleotides, Antisense; C027903:oligo (dT); C013630:oligo (dT) poly (A); D011067:Poly dA-dT; D012313:RNA; D012194:RNA-Directed DNA Polymerase