RNA FISH Protocol for Adherent Cells

RNA fluorescence in situ hybridization (FISH) is a powerful technique that allows for the visualization and localization of specific RNA molecules within individual cells. This method is particularly useful for studying gene expression patterns, RNA trafficking, and the subcellular distribution of RNA transcripts in adherent cell lines.

Fixation of adherent cell lines

• Grow cells on an 18 mm cover glass in a 12-well cell culture plate.
• Aspirate growth medium, and wash with 1 mL of 1×PBS.
• Add 1 mL of fixation buffer.
• Incubate at room temperature for 10 minutes.
• Wash twice with 1 mL of 1×PBS.
• To permeabilize, immerse cells in 1 mL of 70% (vol./vol.) ethanol for at least 1 hour at +2 to +8°C.
• Cells can be stored at +2 to +8°C in 70% ethanol up to a week before hybridization.

Hybridization in adherent cells

If frozen before using, warm the reconstituted probe solution to room temperature. Mix well by vertexing, then centrifuge briefly.

• Aspirate the 70% ethanol off the cover glass containing adherent cells within the 12-well plate.
• Add 1 mL of wash buffer A, and incubate at room temperature for 2-5 minutes.
• Assemble humidified chamber: 150 mm tissue culture plate; bottom lined evenly with a flat water-saturated paper towel and a single layer of Parafilm placed on top of the paper towel. This chamber will help prevent evaporation of the probe solution from under the cover glass.
• Within the humidified chamber, dispense 100 μL of the hybridization buffer containing probe onto the parafilm.
• Gently transfer the cover glass, cells side down, onto the 100 μL drop of hybridization buffer containing probe.
• Cover the humidified chamber with the tissue culture lid, and seal with parafilm.
• Incubate in the dark at 37°C for at least 4 hours. (Incubation can be continued up to 16 hours).
• Gently transfer the cover glass, cells side up, to a fresh 12-well plate containing 1 mL of wash buffer A.
• Incubate in the dark at 37°C for 30 minutes.
• Aspirate the wash buffer A, and then add 1 mL of DAPI nuclear stain to counterstain the nuclei.
• Incubate in the dark at 37°C for 30 minutes.
• Aspirate the DAPI staining buffer, and then add 1 mL of wash buffer B. Incubate at room temperature for 2-5 minutes.
• Add a small drop (5-15 μL depending on the size of the coverslip used) of the mounting medium onto a microscope slide, and mount cover glass onto the slide, cells side down.
• Gently wick away excess anti-fade from the perimeter of the cover glass.
• Seal the cover glass perimeter with clear nail polish, and allow to dry.
• If necessary, gently wipe away any dried salt from the covered glass with water.

Creative Bioarray Relevant Recommendations

• Creative Bioarray offers Custom RNA ISH and RNAscope service. We also offer a range of different FISH services including metaphase and interphase FISH (chromosomal assignment and clone ordering), Fibre-FISH (Chromosome Painting), RNA-FISH (cell-based gene expression assay), M-FISH (multicolor karyotyping), 3D-FISH (on three-dimensionally preserved nuclei), Flow-FISH (quantify the length of telomeres), FISH on paraffin sections (analysis of archive material), ImmunoFISH (combined FISH and IHC).

• When performing RNA FISH, it is imperative to limit RNA degradation. Please ensure that all consumables and reagents are RNase-free.
• When processing multiple samples, cells can be grown in the 12 or 24-well plate or chambered coverslips, keeping the wells properly sealed. Volumes should be adjusted accordingly.

In Situ Hybridization (ISH) & RNAscope Service