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. 2018 Jun 15;8(1):9214.
doi: 10.1038/s41598-018-27547-2.

Reverse Transcription Polymerase Chain Reaction in Giant Unilamellar Vesicles

Mamiko Tsugane  1   2 , Hiroaki Suzuki  3
Affiliations

Reverse Transcription Polymerase Chain Reaction in Giant Unilamellar Vesicles

Mamiko Tsugane et al. Sci Rep. .

Abstract

We assessed the applicability of giant unilamellar vesicles (GUVs) for RNA detection using in vesicle reverse transcription polymerase chain reaction (RT-PCR). We prepared GUVs that encapsulated one-pot RT-PCR reaction mixture including template RNA, primers, and Taqman probe, using water-in-oil emulsion transfer method. After thermal cycling, we analysed the GUVs that exhibited intense fluorescence signals, which represented the cDNA amplification. The detailed analysis of flow cytometry data demonstrated that rRNA and mRNA in the total RNA can be amplified from 10-100 copies in the GUVs with 5-10 μm diameter, although the fraction of reactable GUV was approximately 60% at most. Moreover, we report that the target RNA, which was directly transferred into the GUV reactors via membrane fusion, can be amplified and detected using in vesicle RT-PCR. These results suggest that the GUVs can be used as biomimetic reactors capable of performing PCR and RT-PCR, which are important in analytical and diagnostic applications with additional functions.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Schematic diagram of the RT-PCR system using giant unilamellar vesicles (GUV).
Figure 2
Figure 2
Fluorescence images of GUVs encapsulating RT-PCR mixture and rRNA probes were acquired using laser confocal microscope. The top and bottom images show the GUVs prior and subsequent to thermal cycling (20 and 40 cycles) in the presence and absence of template total RNA, respectively. Red: membrane stained with DiI and Green: Taqman probe (FAM). Scale bar = 10 μm.
Figure 3
Figure 3
(A) Scatter plots of flow cytometric measurements of GUVs containing total RNA and 18S rRNA probes. Horizontal and vertical axes represent the fluorescence intensities of FAM (cDNA amplification) and Alexa 647 (volume marker), respectively. The four conditions correspond to those in Fig. 2 (40 cycles for RT-PCR). (B) Probability distribution plots of IFAM/ITA647 in logarithmic scale for GUVs with V > 65 fL. Plots of all the GUVs (left panel) are segregated and represented as 65 to 524 fL (middle panel) and above 524 fL (right panel) groups.
Figure 4
Figure 4
Fluorescence images of GUVs encapsulating RT-PCR mixture and β-actin probes. The top and bottom images show the contents of GUVs prior and subsequent to thermal cycling (40 cycles) in the presence or absence of template total RNA, respectively. Scale bar = 10 μm.
Figure 5
Figure 5
Fluorescence images of GUVs encapsulating RT-PCR mixture and β-actin probes. The top and bottom images show the contents of GUVs prior and subsequent to thermal cycling (40 cycles) in the presence or absence of synthetic mRNA (0.16 ng/mL), respectively. Scale bar = 10 μm.
Figure 6
Figure 6
Scatter plots of flow cytometric measurements of GUVs obtained by performing amplification using synthetic β-actin mRNA template at various concentrations. Horizontal and vertical axes represent the fluorescence intensities of FAM (cDNA amplification) and Alexa 647 (volume marker), respectively.
Figure 7
Figure 7
(A) Probability distribution plots of IFAM/ITA647 in logarithmic scale for GUVs with V > 65 fL after RT-PCR using various concentrations of mRNA. Plots of all the GUVs (left panel) are segregated and represented as 65 to 524 fL (middle panel) and above 524 fL (right panel) groups. (B) Plot representing the dependence of the relative frequency of reacted liposomes on the concentration of RNA templates.
Figure 8
Figure 8
(A) Schematic representation of electrofusion and RT-PCR experiment. The two populations of GUVs that contain either the enzymes (RT and DNA pol) or template RNA were mixed into a single suspension. After electrofusion, RT-PCR thermal cycling (40 cycles) was conducted in order to amplify the cDNA present solely in the GUVs that contain both the components. (B) Fluorescence images show the GUVs prior and subsequent to electrofusion and after performing RT-PCR. Scale bar = 10 μm.
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