In situ reverse transcription for detection of hybridization between oligonucleotides and their intracellular targets

Joan C. Politz, Robert H. Singer

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)281-285
Number of pages5
JournalMethods: A Companion to Methods in Enzymology
Volume18
Issue number3
DOIs
StatePublished - Jul 1999

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Transcription
Oligonucleotides
Reverse Transcription
Oligodeoxyribonucleotides
RNA
RNA-Directed DNA Polymerase
Assays
Nucleotides
Complementary DNA
Lipids
Messenger RNA
Molecules
oligo (dT)

ASJC Scopus subject areas

  • Molecular Biology

Cite this

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