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Open Access Highly Accessed Methodology article

Visualising single molecules of HIV-1 and miRNA nucleic acids

Kate L Jones1, Adam Karpala23, Bevan Hirst16, Kristie Jenkins3, Mark Tizard3, Cândida F Pereira145, Andrew Leis3, Paul Monaghan3, Alex Hyatt3 and Johnson Mak123*

Author affiliations

1 Centre for Virology, Burnet Institute, Melbourne, Australia

2 School of Medicine, Faculty of Health, Deakin University, Waurn Ponds, Geelong, Victoria 3216, Australia

3 Commonwealth Scientific and Industrial Research Organization, Livestock Industries, Australian Animal Health Laboratory, Geelong, Australia

4 Monash Micro Imaging, Monash University, Clayton, Australia

5 Department of Medicine, Monash University, Clayton, Australia

6 School of Life and Environmental Science, Deakin University, Australia

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Citation and License

BMC Cell Biology 2013, 14:21  doi:10.1186/1471-2121-14-21

Published: 17 April 2013

Abstract

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.

Keywords:
HIV; miRNA; RNA; Oligo-fluorescent in situ hybridization (O-FISH); Visualization; Viral infection