In situ reverse transcription: the magic of strength and anonymity.

Nucleic Acids Research
Anna Ligasová, Karel Koberna

Abstract

In this study, we describe an approach that enables a highly specific, effective and fast detection of polyadenylated RNA sequences in situ at the light and electron microscopy levels. The method developed is based on the incorporation of 5-bromo-2'-deoxyuridine into the growing cDNA strand by means of the reverse transcriptase. We have shown that unlike the previously used deoxyuridine tagged with biotin or digoxigenin, 5-bromo-2'-deoxyuridine is 'invisible' in the DNA-DNA duplex but easily detectable in the DNA-RNA duplex. This feature is an important pre-requisite for the correct interpretation of the data obtained, as our results strongly indicate that reverse transcriptase uses DNA breaks as primers efficiently. We have also shown that the replacement of deoxythymidine by 5-bromo-2'-deoxyuridine considerably stabilizes the growing DNA-RNA duplex, thus enabling the one-step detection of polyadenylated RNA in structurally well-preserved cells. The method developed provides a highly specific signal with the signal/noise ratio higher than 130 for permeabilized cells and 25 for conventional acrylic resin sections under the conditions used. When the high pressure freezing technique followed by the freeze substitution is employed...Continue Reading

References

Jan 1, 1976·Nucleic Acids Research·B R Cullen, M D Bick
Jan 1, 1992·Methods in Enzymology·J EberwineR Finnell
Mar 1, 1990·Proceedings of the National Academy of Sciences of the United States of America·R N Van GelderJ H Eberwine
Feb 25, 1990·Nucleic Acids Research·H MocharlaM E Hodes
Jul 1, 1974·Proceedings of the National Academy of Sciences of the United States of America·J DavidW J Rutter
Aug 24, 1999·Methods : a Companion to Methods in Enzymology·J C Politz, R H Singer

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Methods Mentioned

BETA
light microscopy
electron microscopy

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