Table 2 Advantages and disadvantages of RCA-FISH and smFISH.

Pros

Signal amplification in theory allows to monitor single copy mRNAs also in samples with a high fluorescent background

Specificity: Highly specific probes can be designed to distinguish between paralogues or SNPs

No gene length limitation: short (< 500 bp) genes can be analysed

Modular: Possibility of combination with in situ proximity ligation assays to collect information on mRNA-protein interactions or post-translational modification (not tested in the scope of this paper)

Signal amplification in theory allows to monitor single copy mRNAs in samples with a high fluorescent background

Complexity: easy probe design, few steps minimising contamination risks

Cell permeability: small probes, no large functional molecules involved

Cost of reagents: most reagents needed are standard lab consumables

Time: Fast protocol, ca. 1 day from sample to image, hands on time ca 3 h

Cons

Complexity: many steps increase the risk of RNase contamination or errors

Cell permeability: Involves multiple steps requiring large enzymes which need to reach the cytoplasm. This is a major drawback for use in the plant/algal system

Costs of probes: The expensive LNA- and Padlock probes need to be designed for each gene (ca. 400€ per gene for ~ 200 reactions)

Costs of reagents: pricey reagents and enzymes are used (ca 75€ per reaction)

Time: ca. 2 days from sample to image, of which there are ca 6 h hands on time

Specificity: using standard FISH probes in a “string of beads” manner opens possibility of false positives for isoforms, orthologues or conserved gene families

Gene length limitation: Short mRNA sequences (< 500 bp) are not suitable for smFISH

Costs of probes: 24–48 labelled probes are needed for each gene. Some companies offer discounts on smFISH probes (ca. 500–800€ depending on the number of probes and the fluorophore, ~ 1,000 + reactions)