Document Detail


Massively Parallel Single-Molecule and Single-Cell Emulsion Reverse Transcription Polymerase Chain Reaction Using Agarose Droplet Microfluidics.
MedLine Citation:
PMID:  22455457     Owner:  NLM     Status:  Publisher    
Abstract/OtherAbstract:
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 regents / 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:
Huifa Zhang; Gareth Jenkins; Yuan Zou; Zhi Zhu; Chaoyong James Yang
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Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2012-3-27
Journal Detail:
Title:  Analytical chemistry     Volume:  -     ISSN:  1520-6882     ISO Abbreviation:  -     Publication Date:  2012 Mar 
Date Detail:
Created Date:  2012-3-29     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  0370536     Medline TA:  Anal Chem     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
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