Figure 1.

Single molecule methods of mRNA detection

A – Single molecule FISH (smFISH). 10 probes with 5 fluorophores each are used for mRNA detection with single molecule sensitivity [8]. B – Single molecule inexpensive FISH (smiFISH). The mRNA is detected using non-labelled primary probes, which contain a sequence complementary to target RNA and a read-out sequence identical for all the probes. This read-out sequence hybridizes to a unique secondary probe labelled with 2 fluorophores on its 3ʹ and 5ʹ [13]. C – Sequential smFISH, example of Multiplexed Error Robust FISH (merFISH). RNA targets are first hybridized with encoding probes, containing sequence complementary to target and two read-out sequences. Each RNA target is identified by a combination of uniques read-out sequences in subsequent rounds of hybridization with secondary probes, one secondary probe per round of hybridization. After each round of hybridization with a secondary probe, the signal is detected, registered and the probe is removed. Each RNA is identified with a unique barcode (lower panel, left column), in which a hybridization round functions as a bit. If the RNA gives a signal with a given probe it is assigned 1, in case of no signal it is assigned 0. To increase the sensitivity, sets of 24–100 primary probes are used per mRNA (top panel, left) [16].