The technology uses molecular barcodes and single molecule imaging to detect and count hundreds of unique transcripts in a single reaction without any enzymatic reaction. In brief, a probe library is made with two sequence-specific probes for each gene of interest...
- The first probe is a capture probe that is complementary to a particular target mRNA and contains also a short common sequence coupled to an affinity tag such as biotin.
- The second probe, the reporter probe, contains a second sequence complementary to mRNA and a color-coded tag (with >16000 codes) that provides the detection signal.
- All probes are mixed toghether with total RNA in a single hybridization reaction that results in the formatio of tripartite structures (probe#1-RNA-probe#2).
- After capture on the surface, an applied electric field extends and orients each tripartite structure in the same direction, and then the aligned frame is imaged and analyzed.
- Each target molecule of interest is identified by the color code generated by the ordered fluorescent segments (the barcode) and the level of expression is measured by counting the numbers of codes for each mRNA.
Geiss, G.K., Bumgarner, R.E., Birditt, B., Dahl, T., Dowidar, N., Dunaway, D.L., Fell, H.P., Ferree, S., George, R.D., Grogan, T., James, J.J., Maysuria, M., Mitton, J.D., Oliveri, P., Osborn, J.L., Peng, T., Ratcliffe, A.L., Webster, P.J., Davidson, E.H., Hood, L. (2008). Direct multiplexed measurement of gene expression with color-coded probe pairs. Nature Biotechnology, 26(3), 317-325. DOI: 10.1038/nbt1385