Molecular Beacon Technology for Tissue Engineering Applications

Introduction Detection of RNA in cells is routinely performed by in vitro methods such as RT-PCR and fluorescent in situ hybridization (FISH). RTPCR quantifies gene expression of a cell population. FISH requires fixation of cells, thus unable to give a true picture of mRNA localization. Molecular beacons (MBs) are antisense oligonucleotide stem-loop probes that fluoresce upon hybridization with target nucleic acids. The major advantage of MBs is the ability to detect, localize and monitor specific mRNA expression in single live cells in real-time. However, successful detection of target mRNA by MBs depends on a combination of factors such as target accessibility, efficiency of probe delivery, and the sensitivity of the imaging technique used. Here we report, for the first time, the live-cell imaging of specific mRNA in live cells in a 3D in vitro system using Streptolysin O (SLO) and TAT-peptide conjugation to deliver MBs targeting GAPDH mRNA.