Flow cytometry in combination with fluorescent in situ hybridization (flow-FISH) is
a powerful technique that can be utilized to rapidly detect nucleic acids at single-cell
resolution without the need for homogenization or nucleic acid extraction. Here, we
describe a microfluidic-based method which enables the detection of microRNAs or miRNAs
in single intact cells by flow-FISH using locked nucleic acid (LNA)-containing probes.
Our method can be applied to all RNA species including mRNA and small noncoding RNA
and is suitable for multiplexing with protein immunostaining in the same cell. For
demonstration of our method, this chapter details the detection of miR155 and CD69
protein in PMA and ionomycin-stimulated Jurkat cells. We also include instructions
on how to set up a microfluidic chip sample preparation station to prepare cells for
imaging and analysis on a commercial flow cytometer or a custom-built micro-flow cytometer.