title: Massively parallel single-molecule and single-cell emulsion reverse transcription polymerase chain reaction using agarose droplet microfluidics.
abstract: A microfluidic device for performing single copy, emulsion Reverse Transcription Polymerase Chain Reaction (RT-PCR) within agarose droplets is presented. A two-aqueous-inlet emulsion droplet generator was designed and fabricated to produce highly uniform monodisperse picoliter agarose emulsion droplets with RT-PCR reagents in carrier oil. Template RNA or cells were delivered from one inlet with RT-PCR reagents/cell lysis buffer delivered separately from the other. Efficient RNA/cell encapsulation and RT-PCR at the single copy level was achieved in agarose-in-oil droplets, which, after amplification, can be solidified into agarose beads for further analysis. A simple and efficient method to graft primer to the polymer matrix using 5'-acrydite primer was developed to ensure highly efficient trapping of RT-PCR products in agarose. High-throughput single RNA molecule/cell RT-PCR was demonstrated in stochastically diluted solutions. Our results indicate that single-molecule RT-PCR can be efficiently carried out in agarose matrix. Single-cell RT-PCR was successfully performed which showed a clear difference in gene expression level of EpCAM, a cancer biomarker gene, at the single-cell level between different types of cancer cells. This work clearly demonstrates for the first time, single-copy RT-PCR in agarose droplets. We believe this will open up new possibilities for viral RNA detection and single-cell transcription analysis.
authors.affiliation: State Key Laboratory of Physical Chemistry of Solid Surfaces, The Key Laboratory for Chemical Biology of Fujian Province, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China.
authors.forename: Huifa
authors.initials: H
authors.lastname: Zhang
authors.forename: Gareth
authors.initials: G
authors.lastname: Jenkins
authors.forename: Yuan
authors.initials: Y
authors.lastname: Zou
authors.forename: Zhi
authors.initials: Z
authors.lastname: Zhu
authors.forename: Chaoyong James
authors.initials: CJ
authors.lastname: Yang
mesh_terms: D014408:Biomarkers, Tumor / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D004655:Emulsions; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D006801:Humans; D044085:Microfluidics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012685:Sepharose / Q000737:chemistry*
chemical_list: D014408:Biomarkers, Tumor; D004655:Emulsions; D012685:Sepharose