Fast and accurate quantification of insertion-site specific transgene levels from raw seed samples using solid-state nanopore technology

Abstract

Many modern crop varieties contain patented biotechnology traits, and an increasing number of these crops have multiple (stacked) traits. Fast and accurate determination of transgene levels is advantageous for a variety of use cases across the food, feed and fuel value chain. With the growing number of new transgenic crops, any technology used to quantify them should have robust assays that are simple to design and optimize, thereby facilitating the addition of new traits to an assay. Here we describe a PCR-based method that is simple to design, starts from whole seeds, and can be run to end-point in less than 5 minutes. Subsequent relative quantification (trait vs. non-trait) using capillary electrophoresis performed in 5% increments across the 0–100% range showed a mean absolute error of 1.9% (s.d. = 1.1%). We also show that the PCR assay can be coupled to non-optical solid-state nanopore sensors to give seed-to-trait quantification results with a mean absolute error of 2.3% (s.d. = 1.6%). In concert, the fast PCR and nanopore sensing stages demonstrated here can be fully integrated to produce seed-to-trait quantification results in less than 10 minutes, with high accuracy across the full dynamic range.

Integrins mediate placental extracellular vesicle trafficking to lung and liver in vivo

“…Consistent with our prior results 6 , the concentration of EVs in the plasma of gestation day (GD)14.5 pregnant mice were ~ 2.4-fold higher than in nonpregnant females ( P = 0.015) (Suppl. Figure 1 ).…”

“…Since the discovery of EVs and their effects on distant cells, research interest in the role of placental EVs during pregnancy has greatly increased. The total quantity and concentration of EVs in maternal plasma rise across gestation, with the placenta contributing significantly to this increase 5 , 6 . While a number of effects of placental EVs have been suggested, few studies have attempted to quantify their biodistribution in vivo.…”

“…We sought to address this caveat in multiple ways. First, we used a quantity of EV that mimicked quantities found during pregnancy 6 . Second, we administered EVs into the tail vein, reasoning that this route most closely mimics hematogenous release of placental EV in situ, as both this and the uterine vein ultimately drain into the inferior vena cava.…”

“…EVs were quantified using NanoSight NS300 (Malvern Panalytical,Westborough, MA) as described previously with modifications 6 . Briefly, concentrated samples were diluted 1:500–2000 in PBS and loaded into a 1 ml syringe at a flow rate of 50.…”

“…Because of this intimate association, placental EVs have the potential to communicate with any tissue in the mother that they are able to access. As gestation advances, placental exosome concentration increases in maternal plasma 5 , 6 ; additionally, their bioactivity may change in pathological conditions including preeclampsia and gestational diabetes 7 – 9 . These changes have spurred interest into possible roles of exosomes in regulating immune and cardiovascular responses to pregnancy and parturition.…”