Picoinjection enables digital detection of RNA with droplet rt-PCR

PLoS One. 2013 Apr 26;8(4):e62961. doi: 10.1371/journal.pone.0062961. Print 2013.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line
  • Humans
  • Microfluidic Analytical Techniques / instrumentation
  • Microfluidic Analytical Techniques / methods*
  • Microfluidic Analytical Techniques / statistics & numerical data
  • Microfluidics
  • RNA / analysis*
  • Reverse Transcriptase Polymerase Chain Reaction / instrumentation
  • Reverse Transcriptase Polymerase Chain Reaction / methods*
  • Reverse Transcriptase Polymerase Chain Reaction / statistics & numerical data
  • Sensitivity and Specificity

Substances

  • RNA

Grant support

This work was supported by startup funds from the Department of Bioengineering and Therapeutic Sciences, a Research Award from the California Institute for Quantitative Biosciences (QB3), the Bridging the Gap Award from the Rogers Family Foundation, a New Frontiers Research Award from the UCSF/Sandler Foundation Program for Breakthrough Biomedical Research, and a grant from the University of California Proof of Concept Program. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.