Skip to page navigation menu Skip entire header
Brown University
Skip 13 subheader links

Molecular beacon delivery methods in 2D cell culture

Description

Abstract:
Molecular beacons are small fluorescently labeled strands of DNA that light up when bound to the mRNA of a gene of interest. Delivering molecular beacons to cells is difficult, and current methods lead to excess debris, detrimental background signal, and inaccurate results. This study investigates whether a polymer-based delivery method originally developed for siRNA can be used to deliver molecular beacons with less debris and reduced non-specific background signal than current methods. HEK293 cells were treated with a 1:1 charge ratio of polymer to 35nM beacon complex, then incubated at 37 degrees, fixed with 10% formalin, DAPI stained, and imaged at 20x on a Biotek Cytation 3. Beacon delivery was quantified using a custom MATLAB script to associate the DAPI-stained nuclei with beacon signal above a set threshold for size and signal intensity. Preliminary results show that percent positive cells increased over a 24 hour period for all beacon sequences. At 24 hours, observed percentage of fluorescent cells varied for each beacon sequence. GAPDH (22% positive), quencherless random (17%), quenchered random (11%), and long stem (78%) beacons all displayed fluorescent cells while beacon-only, polymer-only, and no treatment conditions did not. These data indicated that the beacons were successfully delivered into cells, while errant beacon behavior (i.e. low percentage positive cells with quencherless and GAPDH positive control beacons, and high false positive rates with long stem and quenchered random beacons) calls the functionality of these delivered beacons into question, and will require further study. Once these hurdles are overcome, this polymer-based delivery method can be used to study a myriad of cellular processes obscured by the debris and high background signals of currently available delivery methods.

Citation

Holman, Corey, "Molecular beacon delivery methods in 2D cell culture" (2015). Summer Research Symposium. Brown Digital Repository. Brown University Library. https://doi.org/10.26300/j77j-kc47

Relations

Collection:

  • Summer Research Symposium

    Each year, Brown University showcases the research of its undergraduates at the Summer Research Symposium. More than half of the student-researchers are UTRA recipients, while others receive funding from a variety of Brown-administered and national programs and fellowships and go …
    ...