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Visualising single molecules of HIV-1 and miRNA nucleic acids

Jones, Kate L., Karpala, Adam, Hirst, Bevan, Jenkins, Kristie, Tizard, Mark, Pereira, Candida, Leis, Andrew, Monaghan, Paul, Hyatt, Alex and Mak, Johnson 2013, Visualising single molecules of HIV-1 and miRNA nucleic acids, BMC cell biology, vol. 14, no. 21, pp. 1-10, doi: 10.1186/1471-2121-14-21.

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Title Visualising single molecules of HIV-1 and miRNA nucleic acids
Author(s) Jones, Kate L.
Karpala, Adam
Hirst, Bevan
Jenkins, Kristie
Tizard, Mark
Pereira, Candida
Leis, Andrew
Monaghan, Paul
Hyatt, Alex
Mak, JohnsonORCID iD for Mak, Johnson orcid.org/0000-0002-5229-5707
Journal name BMC cell biology
Volume number 14
Issue number 21
Start page 1
End page 10
Total pages 10
Publisher BioMed Central
Place of publication London, England
Publication date 2013
ISSN 1471-2121
Keyword(s) HIV
miRNA
RNA
Oligo-fluorescent in situ hybridization (O-FISH)
Visualization
Viral infection
Summary Background
The scarcity of certain nucleic acid species and the small size of target sequences such as miRNA, impose a significant barrier to subcellular visualization and present a major challenge to cell biologists. Here, we offer a generic and highly sensitive visualization approach (oligo fluorescent in situ hybridization, O-FISH) that can be used to detect such nucleic acids using a single-oligonucleotide probe of 19–26 nucleotides in length.

Results
We used O-FISH to visualize miR146a in human and avian cells. Furthermore, we reveal the sensitivity of O-FISH detection by using a HIV-1 model system to show that as little as 1–2 copies of nucleic acids can be detected in a single cell. We were able to discern newly synthesized viral cDNA and, moreover, observed that certain HIV RNA sequences are only transiently available for O-FISH detection.

Conclusions
Taken together, these results suggest that the O-FISH method can potentially be used for in situ probing of, as few as, 1–2 copies of nucleic acid and, additionally, to visualize small RNA such as miRNA. We further propose that the O-FISH method could be extended to understand viral function by probing newly transcribed viral intermediates; and discern the localisation of nucleic acids of interest. Additionally, interrogating the conformation and structure of a particular nucleic acid in situ might also be possible, based on the accessibility of a target sequence.
Language eng
DOI 10.1186/1471-2121-14-21
Field of Research 110804 Medical Virology
Socio Economic Objective 970111 Expanding Knowledge in the Medical and Health Sciences
HERDC Research category C1 Refereed article in a scholarly journal
Grant ID NHMRC 1025273
Copyright notice ©2013, BioMed Central
Free to Read? Yes
Persistent URL http://hdl.handle.net/10536/DRO/DU:30059964

Document type: Journal Article
Collections: School of Medicine
Open Access Collection
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Every reasonable effort has been made to ensure that permission has been obtained for items included in DRO. If you believe that your rights have been infringed by this repository, please contact drosupport@deakin.edu.au.