8. These probes consist of a pool of oligonucleotides that are
end-labeled with fluorophores. The sequences of these oligonucleotides can be designed on the company website (https://
www.biosearchtech.com/stellaris-designer). Aim to have >20
probes per mRNA transcript. When visualizing short mRNAs,
the limited amount of sequence can limit how many probes are
made. For example with the Sec61β mRNA, which is only
564 nucleotides long, we have worked with as little as
12 probes [18].
9. To ensure that smFISH probes are specific for any given mRNA
target, one can split the pool of Stellaris probes in two, with the
odd and even probes conjugated to different fluorophores, as
we have done previously [11]. This is only possible for transcripts that are stained with a high number of probes (>30).
When the samples are stained with these two pools, all the foci
should be visible in both fluorescent channels. An example is
shown in Fig. 2 where GAPDH is stained with two sets of
probes (odd probes are conjugated to Quazar570, while even
probes are conjugated to Quazar670). In this particular experiment >90% of the foci co-localize. This control can be critical
as many dim foci that are due to either background staining or
autofluorescence may be mistaken for true mRNA-derived
signals. Once this procedure is performed one can estimate
how many foci are expected for that particular mRNA in a
given cell type under a particular condition. We suspect that
many studies involving Stellaris probes overestimate the number of mRNA foci by counting some of these background/
autofluorescent foci signals.
10. Due to the light-sensitive nature of the probes, storing them in
a dark tube or wrapping it in aluminum foil ensures that they
do not lose their fluorescence in storage.
11. We use about 15 μL of mounting solution per 12 mm coverslip. Note that, since the solution is very viscous, it may be
difficult to pipette accurately. Cutting the pipette tip to create a
wider opening (>2 mm in diameter) helps in transferring the
full amount of mounting solution.
12. For optimal digitonin extraction efficiency, as well as visualization by microscopy, one should aim for ~70% confluency prior
to extraction. This typically entails seeding coverslips at ~30%
confluency the day prior to the experiment. Ensure that coverslips are not overgrown, as it makes visualization of individual
cells by microscopy extremely difficult.
13. When blotting excess liquid off with the Kimwipe, we found it
most efficient to tilt the coverslip so that all of the liquid
collects into a droplet—then blotting this droplet with the
Kimwipe by pressing the tissue against the coverslip edge.
Visualizing ER-Associated mRNA
45
end-labeled with fluorophores. The sequences of these oligonucleotides can be designed on the company website (https://
www.biosearchtech.com/stellaris-designer). Aim to have >20
probes per mRNA transcript. When visualizing short mRNAs,
the limited amount of sequence can limit how many probes are
made. For example with the Sec61β mRNA, which is only
564 nucleotides long, we have worked with as little as
12 probes [18].
9. To ensure that smFISH probes are specific for any given mRNA
target, one can split the pool of Stellaris probes in two, with the
odd and even probes conjugated to different fluorophores, as
we have done previously [11]. This is only possible for transcripts that are stained with a high number of probes (>30).
When the samples are stained with these two pools, all the foci
should be visible in both fluorescent channels. An example is
shown in Fig. 2 where GAPDH is stained with two sets of
probes (odd probes are conjugated to Quazar570, while even
probes are conjugated to Quazar670). In this particular experiment >90% of the foci co-localize. This control can be critical
as many dim foci that are due to either background staining or
autofluorescence may be mistaken for true mRNA-derived
signals. Once this procedure is performed one can estimate
how many foci are expected for that particular mRNA in a
given cell type under a particular condition. We suspect that
many studies involving Stellaris probes overestimate the number of mRNA foci by counting some of these background/
autofluorescent foci signals.
10. Due to the light-sensitive nature of the probes, storing them in
a dark tube or wrapping it in aluminum foil ensures that they
do not lose their fluorescence in storage.
11. We use about 15 μL of mounting solution per 12 mm coverslip. Note that, since the solution is very viscous, it may be
difficult to pipette accurately. Cutting the pipette tip to create a
wider opening (>2 mm in diameter) helps in transferring the
full amount of mounting solution.
12. For optimal digitonin extraction efficiency, as well as visualization by microscopy, one should aim for ~70% confluency prior
to extraction. This typically entails seeding coverslips at ~30%
confluency the day prior to the experiment. Ensure that coverslips are not overgrown, as it makes visualization of individual
cells by microscopy extremely difficult.
13. When blotting excess liquid off with the Kimwipe, we found it
most efficient to tilt the coverslip so that all of the liquid
collects into a droplet—then blotting this droplet with the
Kimwipe by pressing the tissue against the coverslip edge.
Visualizing ER-Associated mRNA
45