eliminate nonspecific binding of the probes. This step could be
omitted when the target mRNAs are highly expressed.
5. We usually include hybridizations with a positive control probe
and a negative control probe in each experiment. Any highly
expressed gene or housekeeping gene can be used as a positive
control. A scrambled sequence or a sequence from a gene of a
different species could be used as a negative control. In the
smFISH experiment shown in Fig. 1, we used a mouse
Fig. 1 An example of smFISH imaging in plant tissue. (a) Early-stage maize anthers were hybridized with
probes against the maize gene encoding Dicer-like 5 (DCL5). Green smFISH spots correspond to DCL5
specifically localized to tapetal cell layer. Magenta shows autofluorescence of anther tissue layers. (b) The
green smFISH signal spots of the image in panel (a). (c) Mouse ribosomal protein 17 (MusRpl17) was used as a
negative control. (d) After quantification with Volocity, we detected 97 localization events for DCL5 and 3 for
the negative control. Error bars show standard error. Significance level: <0.05, *; <0.01, **
Single-Molecule FISH Imaging of RNAs in Plants
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