(439g) Using Nanoelectroporation, Molecular Beacons and Single Cell PCR to Quantify the Relationship Between Sirna/MicroRNA Delivery and Gene Silencing In Cancer Cells

Small RNAs such as siRNA and microRNA (miRNA) regulate intracellular pathway in cells and can also serve as a new molecular medicine in cancer therapy
by down- or up-regulation of targeted messenger RNA (mRNA) and proteins. However, our current understanding of the relationship between delivered siRNA/miRNA and
gene silencing in cancer cells is limited because of our inability to deliver the precise dosage and monitor how the delivered siRNA/miRNA affects the individual
cancer cells. The conventional nanoparticle and electroporation based gene delivery methods require a large number of cells and have poor dosage control.
Consequently, the obtained results are an average value from a large cell population and often with poor repeatability.

Recently, we developed a novel nanoelectroporation (NEP) method that can deliver biomolecules such as siRNA, miRNA and molecular beacons (MBs) into individual cells with controlled dosage and real-time monitoring of mRNA and miRNA expression. The precise delivery is achieved by the focused electric field through a nanochannel connecting two microscale channels with a single cell located at the outlet of the nanochannel in one microchannel and detecting/imaging agents located in the other microchannel. By adjusting the magnitude of electric field, pulse length and pulse number, dose of the delivery can be precisely controlled.

Using NEP, we were able to determine the critical dosage of siRNA (Mcl-1) and miR-29b to induce apoptosis of THP-1 (Human acute monocytic leukemia) cell line.The anti-apoptotic
protein Mcl-1 promotes the survival of lymphocytes and hematopoietic stem cells and is linked to drug resistance and poor treatment outcomes in many tumor types. Next, we delivered GAPDH (control) and Mcl-1 MBs into NEP treated cells to detect and monitor the Mcl-1 mRNA expression by confocal microscopy.Finally, we used single-Cell-qRT-PCR to establish the quantitative relationship between the copies of transfected miR-29b and the extent of MCl-1 mRNA down-regulation.

This work is the first to show that the quantitative relationship between siRNA/miRNA delivery and gene silencing/cell apoptosis in cancer cells can be established
using a combination of nanoelectroporation, molecular beacons and single cell qRT-PCR.This analytical technique and resulting information are not only valuable for
better understanding the cell biology but also useful for new drug development.