Single‐Cell Detection of mRNA Expression Using Nanofountain‐Probe Electroporated Molecular Beacons

Abstract

New techniques for single-cell analysis enable new discoveries in gene expression and systems biology. Time-dependent measurements on individual cells are necessary, yet the common single-cell analysis techniques used today require lysing the cell, suspending the cell, or long incubation times for transfection, thereby interfering with the ability to track an individual cell over time. Here a method for detecting mRNA expression in live single cells using molecular beacons that are transfected into single cells by means of nanofountain probe electroporation (NFP-E) is presented. Molecular beacons are oligonucleotides that emit fluorescence upon binding to an mRNA target, rendering them useful for spatial and temporal studies of live cells. The NFP-E is used to transfect a DNA-based beacon that detects glyceraldehyde 3-phosphate dehydrogenase and an RNA-based beacon that detects a sequence cloned in the green fluorescence protein mRNA. It is shown that imaging analysis of transfection and mRNA detection can be performed within seconds after electroporation and without disturbing adhered cells. In addition, it is shown that time-dependent detection of mRNA expression is feasible by transfecting the same single cell at different time points. This technique will be particularly useful for studies of cell differentiation, where several measurements of mRNA expression are required over time.

Micro- and Nanoscale Technologies for Delivery into Adherent Cells

“…Techniques for single-cell localized electroporation have been developed using either glass nanopipettes or microfluidic cantilevers (Figure 2, right column) that are integrated with an electrode to apply the electrical signal through the fluidic channel and are positioned in contact with the membrane of an adhered cell using a micromanipulator [16, 18, 77, 78]. This experimental setup and capability share many similarities to microfluidic nanoinjection including high spatial resolution, dosage control, and versatility for delivering different types and sizes of molecules into adherent cells.…”

“…It is known that stem cells display stochastic behavior within pathways during differentiation due to cross talk between multiple pathways, localization of reactions, and the low concentration of molecules involved in signaling [84–86]. One way to explore such complex stochastic behavior is to integrate unique capabilities of microdevices (i.e., the cell access and analysis module in Figure 3) to include long-term cell culture [3], non-destructive cell access [16, 18, 25, 73], and highly sensitive sensing capabilities [87] with temporal and spatial resolution. Several other microfluidic-based methods, such as isotachophoresis [88], dielectrophoresis [89], and bio-barcodes [90]), could also be integrated for detection and analysis of biosamples by modular assembly.…”

“…The arrayed cell protocol provides a multiplexing capability to conduct many experiments of interest in parallel. The NFP-E has proven to be a versatile technique for molecular delivery to many cell types including immortalized cell lines, stem cells, and immune cells [16, 18]. Successful delivery of proteins, DNA and RNA hairpin molecules, and plasmids have been achieved with high rates of efficiency and viability (>90% for small molecules) into the cytoplasm or the nucleus of the various cells, indicating the broad applicability of the NFP-E system as a robust and versatile biotool [18].…”

Nanoscale bio-platforms for living cell interrogation: current status and future perspectives

“…7E). The system delivered a DNA-based beacon to detect of glyceraldehyde 3-phosphate dehydrogenase and a RNA-based Please do not adjust margins Please do not adjust margins beacon to measure the sequence cloned in the green fluorescence protein mRNA 160 . Moreover, versatile electroporation platforms were also reported for injecting stimulation effectors while detection of the real-time responses.…”

Nanochannel Electroporation as a Platform for Living Cell Interrogation in Acute Myeloid Leukemia

“…Stimulated by demand from biological and medical communities, the development of single‐cell analysis technologies has received broad attention in recent years 1. Unlike established methods for large cell populations, single‐cell analysis technologies generally focus on a small number of cells and can provide differentiation of biological activities at a resolution of intracellular level.…”