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Quantitative assessment of ratiometric bimolecular beacons as a tool for imaging single engineered RNA transcripts and measuring gene expression in living cells.

Zhang X, Song Y, Shah AY, Lekova V, Raj A, Huang L, Behlke MA, Tsourkas A - Nucleic Acids Res. (2013)

Bottom Line: The ability to acquire accurate measurements of RNA copy number in both HT-1080 cells and CHO cells also suggests that RBMBs can be used to image and quantify single RNA transcripts in a wide range of cell lines.Overall, these findings highlight the robustness and versatility of RBMBs as a tool for imaging RNA in live cells.We envision that the unique capabilities of RBMBs will open up new avenues for RNA research.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, University of Pennsylvania, 210 S. 33rd Street, 240 Skirkanich Hall, Philadelphia, PA 19104, USA, Department of Biology, University of Pennsylvania, 433 S. University Ave, 102 Leidy Laboratories, Philadelphia, PA 19104, USA and Integrated DNA Technologies, Inc., 1710 Commercial Park, Coralville, IA 52241, USA.

ABSTRACT
Recently, we developed an oligonucleotide-based probe, ratiometric bimolecular beacon (RBMB), which generates a detectable fluorescent signal in living cells that express the target RNA. Here, we show that RBMBs can also be used to image single RNA transcripts in living cells, when the target RNA is engineered to contain as few as four hybridization sites. Moreover, comparison with single-molecule fluorescence in situ hybridization confirmed that RBMBs could be used to accurately quantify the number of RNA transcripts within individual cells. Measurements of gene expression could be acquired within 30 min and using a wide range of RBMB concentrations. The ability to acquire accurate measurements of RNA copy number in both HT-1080 cells and CHO cells also suggests that RBMBs can be used to image and quantify single RNA transcripts in a wide range of cell lines. Overall, these findings highlight the robustness and versatility of RBMBs as a tool for imaging RNA in live cells. We envision that the unique capabilities of RBMBs will open up new avenues for RNA research.

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Related in: MedlinePlus

Fluorescent images and analysis of HT1080 cells following RBMB delivery and smFISH. HT1080-GFP-96mer cells were microporated in the presence of 0.8 μM RBMBs and placed in the incubator for 5 h. The cells were then fixed and smFISH was performed. Wide-field fluorescent images of (A) GFP, (B) DAPI, (C) RBMB-reference dye (Alexa 750), (D) smFISH probes (TMR) and (E) RBMB-reporter dye (CF640R) were acquired. The images of the smFISH probes and the RBMB reporter dye shown are maximum intensity projections of 56-images within a z-stack. (F) A merged image that includes DAPI (blue) and the maximum intensity projections of smFISH probes (green) and the RBMB-reporter dye (red). A custom Matlab program was used to identify individual spots in the smFISH and RBMB-reporter channels and to calculate the percent colocalization between the two signals. (G) Example Matlab output, showing the smFISH (green circles) and RBMB-reporter (red dots) signals that were detected within the region outlined by a white box in panel F. Spots that were considered to be colocalized are enclosed within a black circle. Notably, the Matlab analysis was performed on the z-stacks (i.e. in three-dimensions) not on the maximum intensity projection images; however, a 2D representation is shown. (H) Overlay of the Matlab output and the merged image shows good correspondence between the two.
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gkt561-F2: Fluorescent images and analysis of HT1080 cells following RBMB delivery and smFISH. HT1080-GFP-96mer cells were microporated in the presence of 0.8 μM RBMBs and placed in the incubator for 5 h. The cells were then fixed and smFISH was performed. Wide-field fluorescent images of (A) GFP, (B) DAPI, (C) RBMB-reference dye (Alexa 750), (D) smFISH probes (TMR) and (E) RBMB-reporter dye (CF640R) were acquired. The images of the smFISH probes and the RBMB reporter dye shown are maximum intensity projections of 56-images within a z-stack. (F) A merged image that includes DAPI (blue) and the maximum intensity projections of smFISH probes (green) and the RBMB-reporter dye (red). A custom Matlab program was used to identify individual spots in the smFISH and RBMB-reporter channels and to calculate the percent colocalization between the two signals. (G) Example Matlab output, showing the smFISH (green circles) and RBMB-reporter (red dots) signals that were detected within the region outlined by a white box in panel F. Spots that were considered to be colocalized are enclosed within a black circle. Notably, the Matlab analysis was performed on the z-stacks (i.e. in three-dimensions) not on the maximum intensity projection images; however, a 2D representation is shown. (H) Overlay of the Matlab output and the merged image shows good correspondence between the two.

Mentions: Although RBMBs appeared to be capable of imaging individual RNA transcripts in live cells, it was unclear whether RBMBs could be used to quantify the total number of RNA transcripts, i.e. measure gene expression, in single cells. To address this question, HT1080-GFP-96mer cells were microporated with various concentrations of RBMBs and fixed 5 h after microporation. smFISH was then performed against the coding sequence of GFP RNA. Single wide-field images of DAPI, GFP and fluorescence from the RBMB reference dye were acquired (Figure 2). In addition, z-stacks of the smFISH and RBMB reporter signals were also acquired. Notably, all dyes are spectrally distinct and confirmed to have little to no bleed-through on our system. An overlay of the maximum intensity projections of smFISH and RBMB reporter images revealed a high degree of colocalization for all RBMB concentrations evaluated between 0.4 µm and 4.8 µm. Representative images are provided in Figure 2. Quantitative analysis in three dimensions, using a custom Matlab algorithm, revealed that ∼71–75% of the RBMB reporter signals colocalized with smFISH signals over this range of RBMB concentrations (Figure 3A). Similarly, ∼70–75% of the smFISH signals colocalized with RBMB reporter signals. Moreover, when the total number of RNA transcripts in each individual cell was counted in the smFISH images and RBMB reporter images, there was nearly a perfect correlation (Figure 3B and Supplementary Figure S1). At RBMB concentrations <0.4 µm, the percent of smFISH signals that colocalized with RBMB reporter signals declined with RBMB concentration. This stemmed from a decrease in the intensity of the RBMB reporter signals, which were often below the fluorescence threshold that was implemented.Figure 2.


Quantitative assessment of ratiometric bimolecular beacons as a tool for imaging single engineered RNA transcripts and measuring gene expression in living cells.

Zhang X, Song Y, Shah AY, Lekova V, Raj A, Huang L, Behlke MA, Tsourkas A - Nucleic Acids Res. (2013)

Fluorescent images and analysis of HT1080 cells following RBMB delivery and smFISH. HT1080-GFP-96mer cells were microporated in the presence of 0.8 μM RBMBs and placed in the incubator for 5 h. The cells were then fixed and smFISH was performed. Wide-field fluorescent images of (A) GFP, (B) DAPI, (C) RBMB-reference dye (Alexa 750), (D) smFISH probes (TMR) and (E) RBMB-reporter dye (CF640R) were acquired. The images of the smFISH probes and the RBMB reporter dye shown are maximum intensity projections of 56-images within a z-stack. (F) A merged image that includes DAPI (blue) and the maximum intensity projections of smFISH probes (green) and the RBMB-reporter dye (red). A custom Matlab program was used to identify individual spots in the smFISH and RBMB-reporter channels and to calculate the percent colocalization between the two signals. (G) Example Matlab output, showing the smFISH (green circles) and RBMB-reporter (red dots) signals that were detected within the region outlined by a white box in panel F. Spots that were considered to be colocalized are enclosed within a black circle. Notably, the Matlab analysis was performed on the z-stacks (i.e. in three-dimensions) not on the maximum intensity projection images; however, a 2D representation is shown. (H) Overlay of the Matlab output and the merged image shows good correspondence between the two.
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gkt561-F2: Fluorescent images and analysis of HT1080 cells following RBMB delivery and smFISH. HT1080-GFP-96mer cells were microporated in the presence of 0.8 μM RBMBs and placed in the incubator for 5 h. The cells were then fixed and smFISH was performed. Wide-field fluorescent images of (A) GFP, (B) DAPI, (C) RBMB-reference dye (Alexa 750), (D) smFISH probes (TMR) and (E) RBMB-reporter dye (CF640R) were acquired. The images of the smFISH probes and the RBMB reporter dye shown are maximum intensity projections of 56-images within a z-stack. (F) A merged image that includes DAPI (blue) and the maximum intensity projections of smFISH probes (green) and the RBMB-reporter dye (red). A custom Matlab program was used to identify individual spots in the smFISH and RBMB-reporter channels and to calculate the percent colocalization between the two signals. (G) Example Matlab output, showing the smFISH (green circles) and RBMB-reporter (red dots) signals that were detected within the region outlined by a white box in panel F. Spots that were considered to be colocalized are enclosed within a black circle. Notably, the Matlab analysis was performed on the z-stacks (i.e. in three-dimensions) not on the maximum intensity projection images; however, a 2D representation is shown. (H) Overlay of the Matlab output and the merged image shows good correspondence between the two.
Mentions: Although RBMBs appeared to be capable of imaging individual RNA transcripts in live cells, it was unclear whether RBMBs could be used to quantify the total number of RNA transcripts, i.e. measure gene expression, in single cells. To address this question, HT1080-GFP-96mer cells were microporated with various concentrations of RBMBs and fixed 5 h after microporation. smFISH was then performed against the coding sequence of GFP RNA. Single wide-field images of DAPI, GFP and fluorescence from the RBMB reference dye were acquired (Figure 2). In addition, z-stacks of the smFISH and RBMB reporter signals were also acquired. Notably, all dyes are spectrally distinct and confirmed to have little to no bleed-through on our system. An overlay of the maximum intensity projections of smFISH and RBMB reporter images revealed a high degree of colocalization for all RBMB concentrations evaluated between 0.4 µm and 4.8 µm. Representative images are provided in Figure 2. Quantitative analysis in three dimensions, using a custom Matlab algorithm, revealed that ∼71–75% of the RBMB reporter signals colocalized with smFISH signals over this range of RBMB concentrations (Figure 3A). Similarly, ∼70–75% of the smFISH signals colocalized with RBMB reporter signals. Moreover, when the total number of RNA transcripts in each individual cell was counted in the smFISH images and RBMB reporter images, there was nearly a perfect correlation (Figure 3B and Supplementary Figure S1). At RBMB concentrations <0.4 µm, the percent of smFISH signals that colocalized with RBMB reporter signals declined with RBMB concentration. This stemmed from a decrease in the intensity of the RBMB reporter signals, which were often below the fluorescence threshold that was implemented.Figure 2.

Bottom Line: The ability to acquire accurate measurements of RNA copy number in both HT-1080 cells and CHO cells also suggests that RBMBs can be used to image and quantify single RNA transcripts in a wide range of cell lines.Overall, these findings highlight the robustness and versatility of RBMBs as a tool for imaging RNA in live cells.We envision that the unique capabilities of RBMBs will open up new avenues for RNA research.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, University of Pennsylvania, 210 S. 33rd Street, 240 Skirkanich Hall, Philadelphia, PA 19104, USA, Department of Biology, University of Pennsylvania, 433 S. University Ave, 102 Leidy Laboratories, Philadelphia, PA 19104, USA and Integrated DNA Technologies, Inc., 1710 Commercial Park, Coralville, IA 52241, USA.

ABSTRACT
Recently, we developed an oligonucleotide-based probe, ratiometric bimolecular beacon (RBMB), which generates a detectable fluorescent signal in living cells that express the target RNA. Here, we show that RBMBs can also be used to image single RNA transcripts in living cells, when the target RNA is engineered to contain as few as four hybridization sites. Moreover, comparison with single-molecule fluorescence in situ hybridization confirmed that RBMBs could be used to accurately quantify the number of RNA transcripts within individual cells. Measurements of gene expression could be acquired within 30 min and using a wide range of RBMB concentrations. The ability to acquire accurate measurements of RNA copy number in both HT-1080 cells and CHO cells also suggests that RBMBs can be used to image and quantify single RNA transcripts in a wide range of cell lines. Overall, these findings highlight the robustness and versatility of RBMBs as a tool for imaging RNA in live cells. We envision that the unique capabilities of RBMBs will open up new avenues for RNA research.

Show MeSH
Related in: MedlinePlus