RNA FISH USING FLUOROPHORE-LABELLED OLIGONUCLEOTIDES (20-mer/Stellaris)
Solutions and material Coverslips (no. 1.5 for best imaging results) 1X PBS pH 7.4 20X PBS pH 7.4 (RNAse-free) DEPC-water 36.5-38% formaldehyde (Sigma F8775, for molecular biology, contains 10-15% methanol) 70% EtOH (dilute 100% EtOH with DEPC-water) Deionized formamide (RNAse-free, store at -20ºC) Microscope slide Fixogum (cement rubber glue) 100mm petri dish 6-well plates 2X SSC 10% formamide/2X SSC 2X Hybridization buffer 4X SSC/ 20% dextran sulfate/0.4% BSA in DEPC-water, store at -20ºC.
Fixative, fresh (3.7% formaldehyde in 1X PBS)
8.5ml DEPC-water + 0.5ml 20X PBS + 1ml formaldehyde 37% Should be RNAse-free, i.e. proteinase K-treated, phenol-extracted, ethanol-precipitated and resuspended at 10mg/ml in RNAse-free water. Store at -20ºC.
200mM Ribonucleoside Vanadyl Complex (e.g. NEB S1402S) DAPI (50l per coverslip)
Add 0.5l of DAPI 0.5mg/ml to 250l of 2X SSC (final conc. 1g/ml)
Prolong Gold (mounting medium from Invitrogen) Protocol
Notes. 1. The hybridization and washing conditions are given for 20-mer oligos/Stellaris probes. If using longer oligos, please see alternative protocol. 2. RNAse-free work! Use clean solutions and wear gloves. 3. 20l of probe hyb mix is needed per 18x18mm coverslip. We typically use oligo probes at a final concentration of 50nM. The optimal probe concentration should be determined empirically. 4. Optimal washing conditions should be determined empirically.
Grow cells on coverslips (I use 18x18mm untreated or gelatinized no. 1.5 coverslips).
Fix 10 minutes at room temperature in freshly made fixative.
Rinse twice with 1X PBS at room temperature.
Wash once in 1X PBS, 3minutes at room temperature.
Transfer the coverslip to 70% EtOH in a 6-well plate. Mix well. Incubate at least overnight at 4°C.
I have found it important to transfer the coverslips to 70% EtOH and not to replace the PBS with ethanol as this will lead to sticking of the coverslip to the bottom of the petri, presumably because of capillary effects. The fixed cells can be stored 2-3 days at 4ºC, but longer storage will result in lower RNA FISH signals.
Assemble as follows 30l of probe hyb mix for each coverslip.
Vortex very vigorously. Mix well. Keep at room temperature.
Rehydrate the coverslips in 2XSSC/10% formamide for at least 15 minutes, at room temperature.
Pipette 20l of probe hyb mix on a microscope slide (do not make air bubbles!).
Take a coverslip with cells. Remove as much liquid as possible. Invert the coverslip with cells on the drop of probe hyb mix. Gently wipe off excess liquid (not too much!).
Seal with Fixogum. Hybridize 4-16 hours at 37°C.
Longer hybridization time is not recommended. POST-HYBRIDIZATION WASHES AND MOUNTING 1.
Gently remove the rubber cement glue around the coverslip. Do not remove coverslip. Care should be taken not to pull on the coverslip during this step.
Transfer the slide to a 100mm petri dish containing 2xSSC and let stand for 2-5 minutes at room temperature.
Gently pull off the coverslip using forceps and transfer to fresh 2X SSC in a 6-well plate, cells facing up.
Wash 30 minutes at 37°C in pre-heated 10% formamide/2XSSC.
These washing conditions are for 20-mer oligos.
Transfer the coverslip to fresh 2X SSC in another 6-well plate. Rinse briefly and change the 2X SSC. Incubate a further 5 minutes in fresh 2XSSC at room temperature.
Stain 2 minutes at room temperature with DAPI, protected from light.
Rinse in 2X SSC and mount with Prolong Gold. Prolong Gold works well with most Stellaris probes. Leave overnight at room temperature to set. Slides should be stored at 4 ºC until and between observation.
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