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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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Quantitative analysis of mRNA copy number in single cells as determined by MP-FISH and LNA-ELF-FISH. Luciferase-MS2 transcripts were fluorescently labeled in HeLa cells by performing MP-FISH and LNA-ELF-FISH, simultaneously. IPLab acquisition software was used to acquire 3D images of both the MP signal (i.e. Cy3) and the ELF signal in 72 randomly selected cells. After 3D deconvolution of the images in IPLab using AutoQuant plug-in software, a 2D image was constructed using a maximum-intensity merged image. The total number of isolated signals was then counted in ImageJ using the particle analysis counter program. The marginal histograms show the distributions of mRNA copy numbers across the population of selected cells as determined by MP-FISH (bottom) and LNA-ELF-FISH (left), respectively.
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Figure 2: Quantitative analysis of mRNA copy number in single cells as determined by MP-FISH and LNA-ELF-FISH. Luciferase-MS2 transcripts were fluorescently labeled in HeLa cells by performing MP-FISH and LNA-ELF-FISH, simultaneously. IPLab acquisition software was used to acquire 3D images of both the MP signal (i.e. Cy3) and the ELF signal in 72 randomly selected cells. After 3D deconvolution of the images in IPLab using AutoQuant plug-in software, a 2D image was constructed using a maximum-intensity merged image. The total number of isolated signals was then counted in ImageJ using the particle analysis counter program. The marginal histograms show the distributions of mRNA copy numbers across the population of selected cells as determined by MP-FISH (bottom) and LNA-ELF-FISH (left), respectively.

Mentions: To assess the specificity of LNA-ELF-FISH, the total number of RNAs (i.e. distinct fluorescent spots) within single cells were counted for 72 cells in both the Cy3 and ELF images. Graphical analysis indicated a linear correlation between the two methods with a slope of 1.05 and R2 value of 0.83 (Figure 2). Both methods were also in close agreement with quantitative RT-PCR data. Specifically, the average RNA copy number per cell as determined by quantitative RT-PCR was 168 ± 15.2, whereas the average RNA copy number per cells as determined by LNA-ELF-FISH and MP-FISH was 126.6 ± 10.7 (SE) and 118.8 ± 9.6 (SE), respectively. A manual transcript-by-transcript comparison between the LNA-ELF and MP-FISH approaches, in six different cells, revealed that 78% of the fluorescent signals in the ELF images co-localized with fluorescent signals in the Cy3 images and 86% of the fluorescent signals in the Cy3 images co-localized with fluorescent signals in the ELF images (Table 1).Figure 2.


Imaging individual microRNAs in single mammalian cells in situ.

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

Quantitative analysis of mRNA copy number in single cells as determined by MP-FISH and LNA-ELF-FISH. Luciferase-MS2 transcripts were fluorescently labeled in HeLa cells by performing MP-FISH and LNA-ELF-FISH, simultaneously. IPLab acquisition software was used to acquire 3D images of both the MP signal (i.e. Cy3) and the ELF signal in 72 randomly selected cells. After 3D deconvolution of the images in IPLab using AutoQuant plug-in software, a 2D image was constructed using a maximum-intensity merged image. The total number of isolated signals was then counted in ImageJ using the particle analysis counter program. The marginal histograms show the distributions of mRNA copy numbers across the population of selected cells as determined by MP-FISH (bottom) and LNA-ELF-FISH (left), respectively.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 2: Quantitative analysis of mRNA copy number in single cells as determined by MP-FISH and LNA-ELF-FISH. Luciferase-MS2 transcripts were fluorescently labeled in HeLa cells by performing MP-FISH and LNA-ELF-FISH, simultaneously. IPLab acquisition software was used to acquire 3D images of both the MP signal (i.e. Cy3) and the ELF signal in 72 randomly selected cells. After 3D deconvolution of the images in IPLab using AutoQuant plug-in software, a 2D image was constructed using a maximum-intensity merged image. The total number of isolated signals was then counted in ImageJ using the particle analysis counter program. The marginal histograms show the distributions of mRNA copy numbers across the population of selected cells as determined by MP-FISH (bottom) and LNA-ELF-FISH (left), respectively.
Mentions: To assess the specificity of LNA-ELF-FISH, the total number of RNAs (i.e. distinct fluorescent spots) within single cells were counted for 72 cells in both the Cy3 and ELF images. Graphical analysis indicated a linear correlation between the two methods with a slope of 1.05 and R2 value of 0.83 (Figure 2). Both methods were also in close agreement with quantitative RT-PCR data. Specifically, the average RNA copy number per cell as determined by quantitative RT-PCR was 168 ± 15.2, whereas the average RNA copy number per cells as determined by LNA-ELF-FISH and MP-FISH was 126.6 ± 10.7 (SE) and 118.8 ± 9.6 (SE), respectively. A manual transcript-by-transcript comparison between the LNA-ELF and MP-FISH approaches, in six different cells, revealed that 78% of the fluorescent signals in the ELF images co-localized with fluorescent signals in the Cy3 images and 86% of the fluorescent signals in the Cy3 images co-localized with fluorescent signals in the ELF images (Table 1).Figure 2.

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