Digital Polymerase Chain Reaction in an Array of Femtoliter Polydimethylsiloxane Microreactors

We developed a simple, compact microfluidic device to perform high dynamic-range digital polymerase chain reaction (dPCR) in an array of isolated 36-femtoliter microreactors. The density of the microreactors exceeded 20 000/mm². This device, made from polydimethylsiloxane (PDMS), allows the samples to be loaded into all microreactors simultaneously. The microreactors are completely sealed through the deformation of a PDMS membrane. The small volume of the microreactors ensures a compact device with high reaction efficiency and low reagent and sample consumption. Future potential applications of this platform include multicolor dPCR and massively parallel dPCR for next generation sequencing library preparation

The <i>Etv5</i> network.

<p>The subnetwork is comprised of <i>Etv5</i> (in lavender in the center), and its differentially-expressed targets (shown in yellow). The remaining central genes, identified by their high betweenness measures relative to the normal network, are shown on the periphery in pink.</p

Expression of Etv5 network in the central nervous system.

<p><b>(A)</b><i>Etv5</i> RNA expression in astrocytes, neurons, OPCs (oligodendrocyte progenitor cells), newly formed oligodendrocytes, myelinating oligodendrocytes, microglia and endothelial cells demonstrates preferential enrichment in astrocytes, OPCs and microglia. Data analyzed from the brain RNA-Seq database [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190001#pone.0190001.ref027" target="_blank">27</a>] (<a href="http://web.stanford.edu/group/barres_lab/brain_rnaseq.html" target="_blank">http://web.stanford.edu/group/barres_lab/brain_rnaseq.html</a>). <b>(B)</b> <i>Gldc</i>, <i>Spry4</i>, <i>Fabp5</i>, <i>Pcdhgc3</i>, <i>Spry2</i>, <i>Shc3</i>, <i>Spred1</i> and <i>Nlgn3</i> expression was enriched in astrocytes, OPCs and microglia. <i>Spred1</i> was the only gene expressed in microglia.</p

Differential expression of central genes identified using the betweenness measure.

<p>Differential expression of central genes identified using the betweenness measure.</p

Percent of target genes (from the regulatory network) that are differentially expressed by each of the central genes identified using the betweenness measure.

<p>Percent of target genes (from the regulatory network) that are differentially expressed by each of the central genes identified using the betweenness measure.</p

Comparison of betweenness measures in the normal and tumor networks.

<p>Filled (red) circles indicate genes whose betweenness measure is at least 1.1 times as large in the tumor network as in the normal network and either a tumor betweenness or normal betweenness value greater than 1e6. These genes are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190001#pone.0190001.t001" target="_blank">Table 1</a>, and shown in pink in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190001#pone.0190001.g004" target="_blank">Fig 4</a>.</p

Thirty-one targets of <i>Etv5</i> are differentially expressed in 3-month-old murine OPG-1 RNA-Seq samples.

<p>Thirty-one targets of <i>Etv5</i> are differentially expressed in 3-month-old murine OPG-1 RNA-Seq samples.</p

“Central genes”: Transcription factors identified as having betweenness values at least 1.1 times as large in the tumor expression network as in the normal expression network and either a tumor betweenness or normal betweenness value greater than 1e6.

<p>“Central genes”: Transcription factors identified as having betweenness values at least 1.1 times as large in the tumor expression network as in the normal expression network and either a tumor betweenness or normal betweenness value greater than 1e6.</p

Summary of differential <i>Etv5</i> expression in additional murine <i>Nf1</i> optic glioma models.

<p>Summary of differential <i>Etv5</i> expression in additional murine <i>Nf1</i> optic glioma models.</p

The <i>Etv5</i> network is differentially expressed in murine <i>Nf1</i> optic gliomas.

<p><i>Gldc</i>, <i>Spry4</i>, <i>Fabp5</i>, <i>Pcdhgc3</i>, <i>Spry2</i>, <i>Shc3</i>, <i>Spred1</i> and <i>Nlgn3</i> RNA expression is increased in optic glioma samples (OPG-1) relative to the normal murine optic nerve (N). n.d., not detected. FC, fold change. p = 0.0003 (<i>Gldc</i>), p = 0.0258 (<i>Spry4</i>), p<0.0001 (<i>Fabp5</i>), p = 0.0005 (<i>Spry2</i>), p = 0.0045 (<i>Shc3</i>), p = 0.0111 (<i>Spred1</i>), p = 0.0008 (<i>Nlgn3</i>). The unadjusted p-values were calculated separately using a t-test.</p