miRNA Detection at Single-Cell Resolution Using Microfluidic LNA Flow-FISH
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.
Key wordsmicroRNA Locked nucleic acid Fluorescence in situ hybridization FISH Flow cytometry Multiplexing Single-cell resolution Microfluidics Rolling circle amplification
The authors would like to thank Dr. Kit S. Lam of UC Davis for insightful discussions, Dr. Aarthi Chandrasakaran for critical reading of the manuscript, and Dr. Chung-yan Koh for providing Qdot reagents. The authors also thank Ron Renzi, JimVan De Vreugde, and Jim He for integration and automation of microfluidic platform. This work was supported by the following grants: R01 DE020891, funded by the NIDCR; the MISL Grand Challenge Laboratory Directed Research and Development program at Sandia National Laboratories; and P50GM085273 (the New Mexico Spatiotemporal Modeling Center) funded by the NIGMS.