34 research outputs found
Evaluation of a Droplet Digital Polymerase Chain Reaction Format for DNA Copy Number Quantification
ABSTRACT: Droplet digital polymerase chain reaction (ddPCR) is a new technology that was recently commercialized to enable the precise quantification of target nucleic acids in a sample. ddPCR measures absolute quantities by counting nucleic acid molecules encapsulated in discrete, volumetrically defined, water-in-oil droplet partitions. This novel ddPCR format offers a simple workflow capable of generating highly stable partitioning of DNA molecules. In this study, we assessed key performance parameters of the ddPCR system. A linear ddPCR response to DNA concentration was obtained from 0.16 % through to 99.6 % saturation in a 20,000 droplet assay corresponding to more than 4 orders of magnitude of target DNA copy number per ddPCR. Analysis of simplex and duplex assays targeting two distinct loci in the Lambda DNA genome using the ddPCR platform agreed, within their expanded uncertainties, with values obtained using a lower density microfluidic chamber based digital PCR (cdPCR). A relative expanded uncertainty under 5 % was achieved for copy number concentration using ddPCR. This level of uncertainty is much lower than values typically observed for quantification of specific DNA target sequences using currently commercially available real-time and digital cdPCR technologies
Straightforward Protein Immobilization Using Redox-Initiated Poly(methyl methacrylate) Polymerization
High-Throughput Microfluidic Single-Cell Digital Polymerase Chain Reaction
Here we present an integrated microfluidic
device for the high-throughput
digital polymerase chain reaction (dPCR) analysis of single cells.
This device allows for the parallel processing of single cells and
executes all steps of analysis, including cell capture, washing, lysis,
reverse transcription, and dPCR analysis. The cDNA from each single
cell is distributed into a dedicated dPCR array consisting of 1020
chambers, each having a volume of 25 pL, using surface-tension-based
sample partitioning. The high density of this dPCR format (118 900
chambers/cm<sup>2</sup>) allows the analysis of 200 single cells per
run, for a total of 204 000 PCR reactions using a device footprint
of 10 cm<sup>2</sup>. Experiments using RNA dilutions show this device
achieves shot-noise-limited performance in quantifying single molecules,
with a dynamic range of 10<sup>4</sup>. We performed over 1200 single-cell
measurements, demonstrating the use of this platform in the absolute
quantification of both high- and low-abundance mRNA transcripts, as
well as micro-RNAs that are not easily measured using alternative
hybridization methods. We further apply the specificity and sensitivity
of single-cell dPCR to performing measurements of RNA editing events
in single cells. High-throughput dPCR provides a new tool in the arsenal
of single-cell analysis methods, with a unique combination of speed,
precision, sensitivity, and specificity. We anticipate this approach
will enable new studies where high-performance single-cell measurements
are essential, including the analysis of transcriptional noise, allelic
imbalance, and RNA processing