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Imaging individual microRNAs in single mammalian cells in situ.

Lu J, Tsourkas A - Nucleic Acids Res. (2009)

Bottom Line: Using this approach, individual miRNAs are identified as bright, photostable fluorescent spots.In this study, miR-15a was quantified in MDA-MB-231 and HeLa cells, while miR-155 was quantified in MCF-7 cells.The dynamic range was found to span over three orders of magnitude and the average miRNA copy number per cell was within 17.5% of measurements acquired by quantitative RT-PCR.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, University of Pennsylvania School of Engineering and Applied Sciences, Philadelphia, PA 19104, USA.

ABSTRACT
MicroRNAs (miRNAs) are potent negative regulators of gene expression that have been implicated in most major cellular processes. Despite rapid advances in our understanding of miRNA biogenesis and mechanism, many fundamental questions still remain regarding miRNA function and their influence on cell cycle control. Considering recent reports on the impact of cell-to-cell fluctuations in gene expression on phenotypic diversity, it is likely that looking at the average miRNA expression of cell populations could result in the loss of important information connecting miRNA expression and cell function. Currently, however, there are no efficient techniques to quantify miRNA expression at the single-cell level. Here, a method is described for the detection of individual miRNA molecules in cancer cells using fluorescence in situ hybridization. The method combines the unique recognition properties of locked nucleic acid probes with enzyme-labeled fluorescence. Using this approach, individual miRNAs are identified as bright, photostable fluorescent spots. In this study, miR-15a was quantified in MDA-MB-231 and HeLa cells, while miR-155 was quantified in MCF-7 cells. The dynamic range was found to span over three orders of magnitude and the average miRNA copy number per cell was within 17.5% of measurements acquired by quantitative RT-PCR.

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Related in: MedlinePlus

Simultaneous detection of individual mRNA molecules using MP-FISH and LNA-ELF-FISH. HeLa cells were engineered to constitutively express luciferase mRNA with 24 MS2 binding repeats in the 3′-untranslated region. Each MS2 site was hybridized by an oligonucleotide probe labeled at its 5′- and 3′-end with Cy3. In addition, the coding region of the luciferase RNA was labeled with a single dig-labeled LNA probe. The LNA probes were subsequently labeled with anti-dig-alkaline phosphatase conjugates and ELF signal amplification was performed. Two-dimensional, deconvolved images of the Cy3 fluorescence, ELF signal and a merged image are shown (top panel). Analogous studies were performed using just Cy3 probes (middle panel) or just LNA probes + ELF amplification (bottom panel). Scale bar, 5 μm.
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Figure 1: Simultaneous detection of individual mRNA molecules using MP-FISH and LNA-ELF-FISH. HeLa cells were engineered to constitutively express luciferase mRNA with 24 MS2 binding repeats in the 3′-untranslated region. Each MS2 site was hybridized by an oligonucleotide probe labeled at its 5′- and 3′-end with Cy3. In addition, the coding region of the luciferase RNA was labeled with a single dig-labeled LNA probe. The LNA probes were subsequently labeled with anti-dig-alkaline phosphatase conjugates and ELF signal amplification was performed. Two-dimensional, deconvolved images of the Cy3 fluorescence, ELF signal and a merged image are shown (top panel). Analogous studies were performed using just Cy3 probes (middle panel) or just LNA probes + ELF amplification (bottom panel). Scale bar, 5 μm.

Mentions: As shown in the top panel of Figure 1, the bright fluorescent spots in the Cy3 image and the ELF image are highly co-localized. The signal elicited by the hybridization of multiple probes to the target RNA was generally not as bright as the ELF signal, particularly in the perinuclear region of most cells, but signal intensity could be improved with the use of more hybridization probes (28). To confirm that the signals in the ELF image did not arise from spectral bleed-through of Cy3 fluorescence into the ELF channel and vice versa, MP FISH and LNA-ELF-FISH were also conducted on separate cell samples (Figure 1, middle and lower panels). No spectral bleed-through was observed in these experiments. Interestingly, the Cy3 signals appeared brighter when MP-FISH was performed independently of LNA-ELF-FISH.Figure 1.


Imaging individual microRNAs in single mammalian cells in situ.

Lu J, Tsourkas A - Nucleic Acids Res. (2009)

Simultaneous detection of individual mRNA molecules using MP-FISH and LNA-ELF-FISH. HeLa cells were engineered to constitutively express luciferase mRNA with 24 MS2 binding repeats in the 3′-untranslated region. Each MS2 site was hybridized by an oligonucleotide probe labeled at its 5′- and 3′-end with Cy3. In addition, the coding region of the luciferase RNA was labeled with a single dig-labeled LNA probe. The LNA probes were subsequently labeled with anti-dig-alkaline phosphatase conjugates and ELF signal amplification was performed. Two-dimensional, deconvolved images of the Cy3 fluorescence, ELF signal and a merged image are shown (top panel). Analogous studies were performed using just Cy3 probes (middle panel) or just LNA probes + ELF amplification (bottom panel). Scale bar, 5 μm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2724290&req=5

Figure 1: Simultaneous detection of individual mRNA molecules using MP-FISH and LNA-ELF-FISH. HeLa cells were engineered to constitutively express luciferase mRNA with 24 MS2 binding repeats in the 3′-untranslated region. Each MS2 site was hybridized by an oligonucleotide probe labeled at its 5′- and 3′-end with Cy3. In addition, the coding region of the luciferase RNA was labeled with a single dig-labeled LNA probe. The LNA probes were subsequently labeled with anti-dig-alkaline phosphatase conjugates and ELF signal amplification was performed. Two-dimensional, deconvolved images of the Cy3 fluorescence, ELF signal and a merged image are shown (top panel). Analogous studies were performed using just Cy3 probes (middle panel) or just LNA probes + ELF amplification (bottom panel). Scale bar, 5 μm.
Mentions: As shown in the top panel of Figure 1, the bright fluorescent spots in the Cy3 image and the ELF image are highly co-localized. The signal elicited by the hybridization of multiple probes to the target RNA was generally not as bright as the ELF signal, particularly in the perinuclear region of most cells, but signal intensity could be improved with the use of more hybridization probes (28). To confirm that the signals in the ELF image did not arise from spectral bleed-through of Cy3 fluorescence into the ELF channel and vice versa, MP FISH and LNA-ELF-FISH were also conducted on separate cell samples (Figure 1, middle and lower panels). No spectral bleed-through was observed in these experiments. Interestingly, the Cy3 signals appeared brighter when MP-FISH was performed independently of LNA-ELF-FISH.Figure 1.

Bottom Line: Using this approach, individual miRNAs are identified as bright, photostable fluorescent spots.In this study, miR-15a was quantified in MDA-MB-231 and HeLa cells, while miR-155 was quantified in MCF-7 cells.The dynamic range was found to span over three orders of magnitude and the average miRNA copy number per cell was within 17.5% of measurements acquired by quantitative RT-PCR.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, University of Pennsylvania School of Engineering and Applied Sciences, Philadelphia, PA 19104, USA.

ABSTRACT
MicroRNAs (miRNAs) are potent negative regulators of gene expression that have been implicated in most major cellular processes. Despite rapid advances in our understanding of miRNA biogenesis and mechanism, many fundamental questions still remain regarding miRNA function and their influence on cell cycle control. Considering recent reports on the impact of cell-to-cell fluctuations in gene expression on phenotypic diversity, it is likely that looking at the average miRNA expression of cell populations could result in the loss of important information connecting miRNA expression and cell function. Currently, however, there are no efficient techniques to quantify miRNA expression at the single-cell level. Here, a method is described for the detection of individual miRNA molecules in cancer cells using fluorescence in situ hybridization. The method combines the unique recognition properties of locked nucleic acid probes with enzyme-labeled fluorescence. Using this approach, individual miRNAs are identified as bright, photostable fluorescent spots. In this study, miR-15a was quantified in MDA-MB-231 and HeLa cells, while miR-155 was quantified in MCF-7 cells. The dynamic range was found to span over three orders of magnitude and the average miRNA copy number per cell was within 17.5% of measurements acquired by quantitative RT-PCR.

Show MeSH
Related in: MedlinePlus