Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

1. Probe Preparation

1. Digest 1 μg of the in vitro transcription vector, pKS(+)pol 236nt ⁹ with Kpn1 enzyme at 37 °C for 1 hour and run the linearized DNA product on an agarose gel. The fragment should be approximately 2500 bp.
2. Cut the fragment out of the gel and purify using the Roche Micro Elute Gel Extraction Kit (all reagents used are listed in Table 1). Perform the final elution in 30 μl elution buffer. Run 5 μl of the product on an agarose gel to verify the purification.
3. Mix an in vitro transcription reaction (see recipe below) using the Roche DIG RNA labeling kit and incubate at 37 °C for 2 hours.

In vitro transcription reaction:

4. Add 228 U of DNase I and incubate at 37 °C for 15 min.
5. Stop the reaction by adding EDTA pH 8.0 to a final concentration of 20 mM.
6. Purify the reaction by using the Quick Spin Columns from Roche as specified by the manufacturer.
7. Purify the probe by precipitation with 1/10 volume DEPC-treated 3M NaOAc pH 5.2 and 2.5 volumes of ice-cold 95% ethanol. Mix and incubate at -80 °C for 30 min to overnight.
8. Centrifuge the probe for 15 min at 4 °C at 15,000 x g.
9. Remove the supernatant and wash the pellet in ice-cold 70% ethanol. Centrifuge again for 5 min at 4 °C at 15,000 x g.
10. Remove the supernatant and dry the pellet. Resuspend the pellet in 50 μl water (DNase/RNase free) containing 10 U Invitrogen RNase OUT.
11. Estimate the concentration of probe by spectrophotometry (measure RNA at 260 nm), and dilute to a concentration of 5 ng/μl. Store in aliquots at -20 °C for short-term use, or -80 °C for long-term storage. It is recommended to perform inter assay comparisons so conserve an aliquot for this.

2. Cell Fixation

1. It is initially important to seed adherent cells onto untreated, sterile glass coverslips so the final confluency upon harvesting is approximately 70-80%. For non-adherent cells, grow as normal and when ready to collect, incubate them with coverslips that have been treated with 0.01% w/v poly-L-lysine (Sigma-Aldrich, Inc.) for 1 hour. For a typical 12-well polystyrene tissue culture plate (3.8 cm²), 150,000 cells (e.g. HeLa) are plated at 24 hours prior to transfection. Non-adherent cells can be infected or transfected as usual and allowed to adhere to glass cover slips before fixation ³.
2. Discard the media and wash the cells with 1X phosphate-buffered saline prepared in double distilled water (DPBS) for 1 min. Diethylpyrocarbonate (DEPC, Sigma-Aldrich, Inc.)-treated 1X PBS may also be used, however untreated PBS cannot as it may contain RNase enzymes.
3. Discard the PBS and add 4% paraformaldehyde, enough to cover the cells completely. Incubate for 15-20 min.
4. Discard the paraformaldeyhyde and wash cells with 1X DPBS for 1 min.
5. Discard the DPBS and add 0.1 M glycine (dissolved in 1X DPBS). Incubate for 10 min.
6. Discard the glycine and wash the cells with 1X DPBS for 1 min.
7. Discard the DPBS and add 0.2% Triton-X (dissolved in 1X DPBS). Incubate for 5-10 min. If staining for nucleolar or nuclear envelope proteins, permeabilize with Triton X-100 for no more than 5 min or the protein localization will become diffuse.
8. Discard Triton X-100 and wash once in 1X DPBS for 1 min.
9. For long-term storage, replace PBS with 70% ethanol and store at 4 °C for up to four months. Alternatively, wash once more with 1X DPBS and proceed to FISH.

3. Fluorescence in situ Hybridization (FISH)

1. If the coverslips were stored in ethanol, replace the ethanol with 1X DPBS and allow the cells to rehydrate for 30 min. Rehydration can be done overnight if the coverslips are stored at 4 °C.
2. Wash the coverslips with 1X DPBS for 1 min.
3. Prepare slides to incubate the coverslips on, taking care to clean and label them well.
4. For an 18mm coverslip, add 50 μl of DNase solution (25 units/coverslip, commercially available) to the slide. Add the coverslip, being sure to place it cell side down, and incubate it for 15 min at room temperature.
5. Wash the coverslips with 1X DPBS for 1 min.
6. Add 50 μl hybridization mix (see recipe below) per coverslip onto a slide and incubate the coverslip cell side down for 16-18 h at 42 °C. The incubation should be performed in a tray containing a 50% formamide, 2X SSPE mix (12.5 ml deionized formamide, 2.5 ml 20X SSPE, 10 ml water).

Hybridization Mix (for one coverslip, 50 μl):

7. Incubate coverslips cell side down in 50 μl 50% formamide (diluted in 1X DPBS) for 15 min at 42 °C.
8. Wash the coverslips twice in 2X SSPE by incubating them cell side down in 50 μl 2X SSPE (20X SSPE diluted in 1X DPBS) for 5 min each at 42 °C.
9. Wash the coverslips in 1X DPBS for 1 min.

4. Immunofluorescence Staining

1. Block the coverslips by placing them cell side down in 50 μl 1X Roche blocking solution (10X Roche blocking solution diluted in 1X DPBS) for 30 min.
2. Incubate the coverslips cell side down in 50 μl primary antibody solution for 1 hour at 37 °C. Anti-DIG antibody should be diluted according to manufacturer’s direction in 1X blocking solution. If other antibodies are being used to stain proteins, they may be added at the correct concentration provided that all antibodies used are derived from different host species.
3. Wash the coverslips for 10 min in 1X DPBS.
4. Incubate the coverslips cell side down in 50 μl secondary antibody solution for 1 hour at 37 °C. Alexa Fluor-conjugated antibodies (Invitrogen) can be used to generate a range of colors and are used at 1:500 in 1X blocking solution, 1X DPBS.
5. Wash the coverslips twice for 10 min each in 1X DPBS.
6. Dry the coverslips cell side up on Whatman paper. Be sure to cover the coverslips to avoid exposure to light and bleaching.
7. Once all liquid is evaporated, mount the coverslips onto fresh slides using 8 μl ImmunoMount (Thermo Scientific, Inc.). Gently tap down the coverslip to eliminate any air bubbles.
8. Apply nail polish to the edges of the coverslip to secure it in place.
9. The visualization of RNA and proteins by microscopy techniques is also critical to the success of this technique. Settings on the microscope used can help or hinder visualization so it is key that the settings on the microscope be set correctly and be consistent from one sample to another in a given experiment. For detailed microscopy settings, optics and antibody combinations please see references^1,10.

5. Representative Results

An example of viral RNA staining can be seen in Figure 2. The viral RNA for HIV-1 is seen throughout the cytoplasm displayed diffusely for the most part, although small cytoplasmic punctae are not uncommon. The specificity can be seen by comparing positive cells to surrounding cells that display no fluorescence. As mentioned in the introduction, HIV-1 that lacks the regulatory Rev protein produces RNA which is retained in the nucleus: this can be visualized as a bright signal in the nucleus, and a lack of RNA fluorescence signal in the cytoplasm. Overexpression of cellular proteins is also capable of shifting the distribution of the HIV-1 viral RNA. In Figure 2, the overexpression of proteins involved in endosomal vesicle trafficking (e.g., Rab7-interacting lysosomal protein (RILP)¹¹ or N-terminally deleted RILP (RILPN)¹¹, and proteins that interfere with dynein motor function [e.g. p50/dynamitin^1,12], interfere with the normal steady-state localization of the viral genomic RNA as determined by FISH analyses. RILP expression leads to re-localization of the viral genomic RNA to the microtubule organization center (MTOC) ¹³; RILPN disperses late endosomes within the cytoplasm due to the inability to bind to p150^Glued of the dynein motor complex ¹⁴ and p50/dynamitin blocks the large subunit of dynein motor and releases late endosomes but also the viral genomic RNA and HIV-1 structural proteins to the cell periphery¹ (Figure 2). The manipulation of the steady-state localization of the viral genomic RNA, a key contributor to the infectiousness of virus particles, would be key to the eventual development of therapeutics. In our hands, viral RNA appears in the cell as early as 3 hours post-transfection and infection¹⁰ and becomes readily observable by this FISH technique by 12 hours.

Figure 1. Flow chart of the protocol for FISH/IF co-analyses. Cells are grown on coverslips and then fixed with paraformaldehyde. Treatments of glycine and Triton-X are performed before the cells are either used for FISH/IF or stored in 70% ethanol for storage. Cells dehydrated for storage are rehydrated in 1X DPBS (DEPC-treated PBS) before continuing on to FISH/IF. Cells are treated with DNase I and left overnight to hybridize with probe. Once hybridization is complete, the cells are washed with formamide, as well as with SSPE, and a final 1X DPBS rinse. Blocking solution is applied to the cells, followed by incubation with the primary antibodies. After washing, secondary antibodies are applied. Two final washes in 1X DPBS complete the staining procedure where upon the coverslips are dried and mounted on slides for imaging.

Figure 2. Representative results using FISH/IF co-analyses. A) HIV-1 is transfected into HeLa cells and in some cases with constructs that cause the overexpression of cellular proteins (RILP, p50/Dynamitin and RILPΔN (an amino-terminal deletion mutant)). FISH/IF co-analyses was performed: RNA is identified in green with either G3BP or LAMP1 (red) to differentiate. B) HIV-1 is expressed in HeLa and collected at different time points. Viral RNA expression is tracked in green while the cellular protein, hnRNP A1, is stained in red. Size bars are 10 μm. Images modified from references ¹ (select figures from panel A; RILP, p50/Dynamitin, and RILP deltaN) and ¹⁷ (panel B).