title: Picoinjection enables digital detection of RNA with droplet rt-PCR.
abstract: The ability to add reagents to drops in a sequential fashion is necessary for numerous applications of microfluidics in biology. An important method for accomplishing this is picoinjection, a technique in which reagents are injected into aqueous drops using an electric field. While picoinjection has been shown to allow the precise addition of reagents to drops, its compatibility with biological reactions is yet to be thoroughly demonstrated. Here, we investigate the compatibility of picoinjection with digital RT-PCR Taqman assays, reactions that incorporate nucleic acids, enzymes, and other common biological reagents. We find that picoinjection is compatible with this assay and enables the detection of RNA transcripts at rates comparable to workflows not incorporating picoinjection. We also find that picoinjection results in negligible transfer of material between drops and that the drops faithfully retain their compartmentalization.
authors: Department of Bioengineering and Therapeutic Sciences, California Institute for Quantitative Biosciences, University of California San Francisco, San Francisco, California, USA.
authors: Dennis J
authors: DJ
authors: Eastburn
authors: Adam
authors: A
authors: Sciambi
authors: Adam R
authors: AR
authors: Abate
mesh_terms: D002460:Cell Line; D006801:Humans; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods* / Q000706:statistics & numerical data; D044085:Microfluidics; D012313:RNA / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods* / Q000706:statistics & numerical data; D012680:Sensitivity and Specificity
chemical_list: D012313:RNA
references: Proc Natl Acad Sci U S A. 2010 Nov 9;107(45):19163-6
references: 20962271
references: Lab Chip. 2008 Feb;8(2):198-220
references: 18231657
references: Chem Biol. 2008 May;15(5):427-37
references: 18482695
references: Lancet. 1964 Feb 1;1(7327):238-40
references: 14086209
references: Lab Chip. 2006 Mar;6(3):437-46
references: 16511628
references: Anal Chem. 2008 Dec 1;80(23):8975-81
references: 19551929
references: Invest Urol. 1979 Jul;17(1):16-23
references: 447482
references: Rep Prog Phys. 2012 Jan;75(1):016601
references: 22790308
references: Proc Natl Acad Sci U S A. 2009 Aug 25;106(34):14195-200
references: 19617544
references: Anal Chem. 2004 Sep 1;76(17):4977-82
references: 15373431
references: Angew Chem Int Ed Engl. 1998 Mar 16;37(5):550-575
references: 29711088
references: Anal Chem. 2008 Mar 15;80(6):1854-8
references: 18278951
references: Angew Chem Int Ed Engl. 2006 Nov 13;45(44):7336-56
references: 17086584
references: Electrophoresis. 2005 Oct;26(19):3706-15
references: 16136526
references: Lab Chip. 2012 Oct 21;12(20):4029-32
references: 22930333
references: Proc Natl Acad Sci U S A. 2010 Mar 2;107(9):4004-9
references: 20142500
references: J Biosci Bioeng. 2005 Mar;99(3):293-5
references: 16233792
references: Phys Rev Lett. 2001 Apr 30;86(18):4163-6
references: 11328121