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.
Highlights
The biological counterpart of the boundary of minute reaction environment is the lipid bilayer, which is ubiquitously present in living cells owing to its ability to form functional interfaces in an aqueous environment, adaptability to multiscales, and selective transportation of various chemical species and cargos
During reverse transcription polymerase chain reaction (RT-PCR), initially the cDNAs are synthesised from the RNA with reverse transcriptase and these cDNAs are amplified using PCR
We demonstrated that the reverse transcription and amplification of transcripts using the total RNA as well as synthetic mRNA could be conducted in giant unilamellar vesicles (GUVs)
Summary
The biological counterpart of the boundary of minute reaction environment is the lipid bilayer, which is ubiquitously present in living cells owing to its ability to form functional interfaces in an aqueous environment, adaptability to multiscales, and selective transportation of various chemical species and cargos. Shohda et al.[32] performed PCR of 1229 bp DNA harbouring green fluorescent protein (GFP) gene in giant multilamellar vesicles (MLVs) obtained by the freeze-dried empty liposome method They found that the reaction efficiency was ~20% and ~80% in 2.7 μm (10 fL) and 10 μm (~500 fL) vesicles, respectively. We are developing a microreactor system using giant unilamellar vesicles (GUVs), which consists of single lipid bilayer to the plasma membrane of cells, obtained by the water-in-oil (W/O) emulsion transfer method This method was originally developed by Pautot et al.[35], and can produce unilamellar vesicles of sizes up to ~100 μm in diameter[36], and exhibits 100% encapsulation efficiency for a large range of molecular sizes and concentrations of reagents. The serial dilution experiment of the template synthetic mRNA proved that the RT-PCR was successfully conducted using a small number of RNA molecules
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