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Single‐Cell Detection of mRNA Expression Using Nanofountain‐Probe Electroporated Molecular Beacons
Author(s) -
GiraldoVela Juan P.,
Kang Wonmo,
McNaughton Rebecca L.,
Zhang Xuemei,
Wile Brian M.,
Tsourkas Andrew,
Bao Gang,
Espinosa Horacio D.
Publication year - 2015
Publication title -
small
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.785
H-Index - 236
eISSN - 1613-6829
pISSN - 1613-6810
DOI - 10.1002/smll.201401137
Subject(s) - molecular beacon , electroporation , transfection , microbiology and biotechnology , messenger rna , oligonucleotide , cell , biology , single cell analysis , gene knockdown , gene expression , cell culture , gene , genetics
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.