DNA and RNA FISH Protocol: Sample Preparation, Hybridisation and Imaging

ABMIUM laboratory protocol

A complete DNA and RNA fluorescence in situ hybridisation protocol covering sample preparation, fixation, permeabilisation, probe hybridisation, washing, imaging and troubleshooting.

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Fluorescence in situ hybridisation (FISH) enables the spatial localisation of specific nucleic acid sequences within intact cells or tissue sections using labelled oligonucleotide or polynucleotide probes. Combined DNA and RNA FISH extends this principle to allow simultaneous or sequential interrogation of genomic loci and their transcriptional output within the same specimen, providing powerful spatiotemporal information about gene expression and chromosomal organisation.

RNA detection by FISH requires that cellular RNA integrity is preserved throughout fixation, permeabilisation, and hybridisation. Conversely, DNA FISH requires denaturation of double-stranded genomic DNA, which presents a risk to RNA stability if performed simultaneously. The sequential approach described in this protocol, in which RNA FISH is performed prior to DNA denaturation, is the most widely used strategy for combined applications and is supported by the published literature on simultaneous detection of nascent RNA and chromosomal loci.

This protocol is intended as a generic framework. Probe design, hybridisation conditions, stringency wash parameters, and signal-to-noise optimisation are target- and probe-dependent and must be validated for each experimental system.

Equipment required

Fluorescence microscope equipped with appropriate filter sets for the fluorophores in use (widefield configuration)

Humidified hybridisation chamber (light-tight; commercially available or fabricated from a sealed slide box lined with damp tissue)

Water bath or heat block capable of maintaining 37 degrees C, 73 to 80 degrees C, and intermediate temperatures with accuracy of plus or minus 1 degree C

Coplin jars or slide staining troughs (RNase-free; dedicate separate sets for RNA and DNA steps)

Precision forceps and coverslip handling tools

Microcentrifuge and benchtop centrifuge

Vortex mixer

pH meter calibrated with appropriate buffers

Timer

UV crosslinker (optional; for additional RNase decontamination of surfaces and plasticware)

Reagents

Fixation and Permeabilisation

16% methanol-free paraformaldehyde (PFA), diluted to working concentration in phosphate-buffered saline (PBS) immediately before use

Phosphate-buffered saline (PBS), pH 7.4, prepared RNase-free

Triton X-100 or NP-40 (non-ionic detergent for permeabilisation; concentration is sample-dependent and must be optimised)

For FFPE sections: xylene or equivalent clearing agent, graded ethanol series, antigen retrieval reagent (target retrieval solution or citrate buffer, pH 6.0; selection is target-dependent)

RNase-Free Handling Reagents

DEPC-treated water or commercially supplied RNase-free water (confirm RNase-free status by manufacturer certificate or RNaseAlert assay)

RNase decontamination solution (for bench surfaces, pipettes, and non-autoclavable equipment)

RNase inhibitor (optional; may be included in hybridisation buffers for sensitive targets)

Hybridisation and Stringency Wash Reagents

20x SSC stock solution (sodium chloride / sodium citrate, pH 7.0): prepare RNase-free; autoclave or filter-sterilise

Deionised formamide (deionised to remove charged breakdown products; store at minus 20 degrees C in aliquots under nitrogen if possible; treat as a hazardous material)

Blocking reagents as appropriate to probe and detection chemistry (e.g., salmon sperm DNA, COT-1 DNA, dextran sulfate, Denhardt's solution; inclusion and concentration are probe- and target-dependent)

Hybridisation buffer: composition is probe- and target-class-dependent; a commonly used formulation for RNA FISH contains 50% formamide, 2x SSC, 10% dextran sulfate, and blocking DNA; exact composition must be validated per probe set

Wash buffers: 2x SSC, 1x SSC, and 0.1x SSC prepared in RNase-free water; exact stringency is probe- and target-dependent

DNA Denaturation Step

70% formamide in 2x SSC (for standard chromosomal DNA FISH denaturation): prepare fresh; pH must be confirmed at 7.0 plus or minus 0.2 prior to use

Graded ethanol series (70%, 90%, 100%) pre-chilled to minus 20 degrees C for post-denaturation dehydration

Counterstain and Mounting

DAPI (4',6-diamidino-2-phenylindole) working solution: concentration is typically in the range of 100 ng/mL to 1 microgram/mL in PBS or 2x SSC; must be optimised to avoid bleed-through into probe detection channels

Antifade mounting medium compatible with aqueous preparations and validated for the fluorophores in use; refractive index should be appropriate for the objective in use

Clear nail varnish or optical adhesive for coverslip sealing

Probes

Directly labelled fluorescent probes for both DNA and RNA targets. Selection of fluorophore, probe format (oligonucleotide, BAC-derived, synthetic), and labelling density must be matched to the detection sensitivity required for the target, the microscopy platform, and the spectral properties of the filter set. Probe validation (specificity, efficiency, and absence of off-target signal) must be performed prior to experimental use. Probe working concentrations are probe-dependent and must be determined empirically.

RNase-Free Environment and RNA Integrity

Preservation of cellular RNA is the single most critical pre-analytical requirement for combined DNA and RNA FISH. RNases are ubiquitous, thermostable, and highly active at room temperature. Contamination introduced at any point prior to hybridisation can degrade RNA targets and produce false-negative or unreliable RNA FISH signals. The following practices must be observed throughout all RNA-related steps.

Workspace Preparation

Decontaminate all bench surfaces, pipettors, and non-autoclavable equipment with an RNase decontamination solution before beginning. Allow sufficient contact time as directed for the product in use.

Prepare a dedicated RNA workspace, physically separated from areas where RNase-containing reagents (e.g., DNase/RNase solutions, gel reagents) are routinely handled.

UV crosslink the cleaned bench surface and any reusable plasticware if a UV crosslinker is available (254 nm, 1 to 2 J/cm squared; refer to crosslinker manufacturer's guidance).

Use only RNase-free certified consumables: tubes, pipette tips, Coplin jars, and coverslip-handling equipment. Do not reuse consumables between experiments without thorough decontamination.

Wear powder-free nitrile gloves throughout all RNA steps. Change gloves frequently, particularly after touching the face, hair, or non-decontaminated surfaces.

Reagent Preparation

Prepare all aqueous solutions used during RNA FISH steps with DEPC-treated or certified RNase-free water.

Autoclave buffers that can withstand autoclaving (e.g., PBS, SSC). Filter-sterilise buffers that contain reagents incompatible with autoclaving.

Aliquot RNase-free reagents into single-use volumes where possible to prevent iterative freeze-thaw contamination risk.

RNA Integrity Controls

A dedicated RNA integrity control should be included in every combined FISH experiment. This serves both to confirm that RNA targets are preserved through the protocol and to identify any step at which RNA degradation may have occurred.

Positive control: a well-characterised, abundantly expressed RNA target (e.g., a housekeeping transcript) that should consistently produce signal in the sample type under investigation.

Negative control for RNA specificity: an RNase A pre-treatment control. Treat a subset of slides or coverslips with RNase A (concentration and conditions should be validated to ensure complete degradation of the target RNA class) prior to hybridisation. Complete loss of RNA FISH signal in these samples confirms probe specificity and validates RNA detection.

No-probe control: process a sample through the complete protocol without addition of the RNA probe. This establishes the autofluorescence baseline and assesses non-specific probe-independent signal.

Sample Preparation

Cultured Cells on Coverslips

Cells should be plated on coverslips (glass, appropriate thickness for the objective in use; number one and a half, 0.17 mm, is appropriate for most high-numerical-aperture objectives) at a density that achieves 60 to 80% confluency at the time of fixation. Overcrowding reduces signal quality and complicates image analysis. Coverslips should be pre-cleaned and, where required, coated with an appropriate cell adhesion substrate to prevent cell detachment during permeabilisation and denaturation steps.

Aspirate the culture medium and wash cells gently with pre-warmed PBS (37 degrees C). Avoid allowing cells to dry at any point during washing.

Fix cells immediately with freshly prepared PFA at the working concentration in PBS. The PFA concentration must be empirically validated for the cell type and the targets of interest; a range of 2% to 4% (w/v) is commonly used for combined FISH but is not universally established across all cell types and probe chemistries.

Incubate at room temperature for 10 to 15 minutes with gentle rocking. Longer fixation times can reduce probe accessibility and RNA signal; shorter times may be insufficient for DNA FISH.

Wash with PBS (three times, 5 minutes each, room temperature) to remove residual PFA.

Permeabilise cells with a detergent solution in PBS. The detergent type, concentration, and incubation time are cell-type-dependent and must be optimised. Excessive permeabilisation can result in loss of cellular morphology and nuclear content; insufficient permeabilisation reduces probe penetration.

Wash with PBS (three times, 5 minutes each, room temperature).

Proceed directly to Section 5 (RNA FISH) or store coverslips in 70% ethanol at minus 20 degrees C for short-term storage (up to several weeks). Note: storage in ethanol will dehydrate the sample; rehydrate through a graded ethanol series before proceeding.

Tissue Sections (FFPE)

FFPE tissue processing introduces extensive cross-linking that reduces probe accessibility for both DNA and RNA targets. Antigen and nucleic acid retrieval is required, and conditions must be validated for the target and tissue type. RNA preservation in FFPE material is variable and depends on pre-analytical factors including fixation time, formalin concentration, and processing conditions. It is strongly recommended to confirm RNA integrity in FFPE samples using a validated surrogate assay (e.g., short amplicon RT-PCR or RNA quality scoring from extracted RNA) before committing to combined FISH.

Section FFPE tissue blocks at 4 to 6 micrometres onto positively charged glass slides. Bake sections at 60 degrees C for 30 to 60 minutes to promote adhesion.

Deparaffinise sections through xylene (or equivalent; two changes, 10 minutes each) followed by a graded ethanol series (100%, 100%, 90%, 70%; 2 minutes each) and rehydration to water.

Perform heat-induced nucleic acid retrieval using an appropriate retrieval solution preheated to near-boiling temperature. The retrieval buffer composition, pH, temperature, and duration are tissue-, fixation-, and target-dependent and must be optimised. Allow slides to cool to room temperature in the retrieval solution, then wash in PBS.

Treat sections with proteinase K (concentration, temperature, and incubation time are tissue- and fixation-dependent and must be empirically optimised) to improve probe penetration. Under-digestion reduces signal; over-digestion destroys tissue morphology.

Wash with PBS (two to three times), then post-fix in 4% PFA in PBS for 5 to 10 minutes to stabilise tissue structure after protease treatment.

Wash with PBS (three times, 5 minutes each), then proceed to Section 5.

Tissue Sections (Fresh-Frozen Cryosections)

Fresh-frozen cryosections generally offer superior RNA preservation compared to FFPE. However, the cross-linking chemistry available in frozen sections is limited, and structural preservation may be less robust during the denaturing steps required for DNA FISH. The fixation and permeabilisation conditions below should be treated as a starting framework and optimised for the specific tissue.

Section frozen tissue at 5 to 10 micrometres and mount directly onto positively charged or poly-L-lysine coated glass slides. Air-dry sections briefly (no more than 30 to 60 seconds; avoid prolonged drying, which degrades RNA).

Fix in 4% PFA in PBS for 10 to 15 minutes at room temperature.

Wash with PBS (three times, 5 minutes each).

Permeabilise with 0.1% to 0.5% Triton X-100 in PBS; concentration and duration are tissue-dependent and must be optimised.

Wash with PBS (three times, 5 minutes each), then proceed to Section 5.

RNA FISH: Hybridisation and Detection

RNA FISH is performed prior to DNA denaturation in the combined protocol. This sequential order protects RNA targets from the alkaline and thermal conditions used for DNA denaturation, which would otherwise compromise RNA integrity and RNA FISH signal.

Pre-Hybridisation

Equilibrate samples in 2x SSC for 5 minutes at room temperature.

Incubate samples in pre-hybridisation buffer (composition is probe- and target-dependent; typically 50% formamide in 2x SSC or a proprietary equivalent) for 30 to 60 minutes at the hybridisation temperature. This step reduces non-specific probe binding.

Probe Preparation and Hybridisation

Prepare the RNA probe hybridisation mixture according to the validated probe protocol. Direct fluorophore-labelled RNA probes should be diluted in hybridisation buffer to the empirically validated working concentration. Denature the probe mixture as required by probe type and format (conditions are probe-dependent).

Remove samples from the pre-hybridisation buffer. Do not allow samples to dry.

Apply the probe hybridisation mixture to the sample. Use a volume sufficient to cover the entire sample area uniformly. Cover with a glass coverslip or parafilm strip to prevent evaporation.

Incubate in a humidified, light-tight chamber. Hybridisation temperature and duration are probe- and target-dependent. Many RNA FISH probe formats use a hybridisation temperature in the range of 37 to 42 degrees C for 4 to 16 hours, but this must be validated.

Post-Hybridisation Washes

Stringency washes remove unbound and non-specifically bound probe. Wash stringency (SSC concentration, formamide concentration, temperature, and wash duration) is probe- and target-dependent. Insufficient stringency produces background signal; excessive stringency reduces specific signal. The following represents a general starting framework and must be optimised.

Remove the coverslip or parafilm carefully, avoiding disruption of the sample.

Wash in 2x SSC at the hybridisation temperature for 5 minutes (two times). This removes the bulk of unbound probe.

Wash in 1x or 0.5x SSC (with or without formamide, as appropriate for the probe; conditions are probe-dependent) at the hybridisation temperature for 5 to 10 minutes.

Wash in 2x SSC at room temperature for 5 minutes.

Briefly rinse in PBS.

After post-hybridisation washes, RNA FISH signal should be confirmed by imaging before proceeding to DNA denaturation if the combined procedure is performed on the same physical sample. If signal quality is unsatisfactory, do not proceed; troubleshoot the RNA FISH step first.

DNA FISH: Denaturation, Hybridisation, and Detection

DNA FISH requires denaturation of the double-stranded genomic DNA target to allow probe hybridisation. This denaturation step, particularly the combined denaturation of sample and probe at high temperature in formamide-containing buffers, is the step most likely to compromise RNA integrity if RNA FISH has been performed first. Where preserving RNA FISH signal post-DNA denaturation is required, the denaturation conditions should be kept to the minimum necessary to achieve adequate DNA FISH signal, and conditions should be validated in pilot experiments.

Sample Denaturation

Dehydrate samples through an ice-cold graded ethanol series (70%, 90%, 100%; 2 minutes each) immediately before denaturation. This step is critical for chromosomal DNA FISH and helps stabilise the sample structure. Air-dry briefly (30 to 60 seconds).

Denature the sample in 70% formamide in 2x SSC at 73 degrees C for 2 to 5 minutes. Temperature, time, and formamide concentration are probe- and target-dependent and must be optimised for the specific DNA target. Over-denaturation destroys nuclear morphology; under-denaturation results in absence of DNA FISH signal.

Immediately dehydrate through ice-cold graded ethanol series (70%, 90%, 100%; 2 minutes each) to terminate denaturation rapidly and fix the denatured state. Air-dry briefly.

DNA Probe Preparation and Hybridisation

Prepare the DNA probe hybridisation mixture. Directly labelled DNA probes should be diluted to the empirically validated working concentration in an appropriate hybridisation buffer (commonly containing 50% formamide, 2x SSC, 10% dextran sulfate, and blocking DNA such as COT-1 DNA or salmon sperm DNA; exact composition is probe- and target-dependent).

Denature the DNA probe mixture separately according to the validated probe protocol (typically 75 to 80 degrees C for 5 to 10 minutes, followed by pre-annealing at 37 degrees C for 30 to 60 minutes if blocking of repetitive sequences is required; conditions are probe-dependent).

Apply the denatured probe mixture to the dehydrated sample. Cover with a glass coverslip or parafilm. Seal the edges with rubber cement or equivalent to prevent evaporation during the overnight hybridisation.

Hybridise in a humidified, light-tight chamber at 37 degrees C overnight (typically 12 to 16 hours). Hybridisation time and temperature are probe-dependent.

Post-Hybridisation Washes

Remove rubber cement sealant and allow the coverslip to float off in 2x SSC at 37 to 42 degrees C (do not force removal).

Wash in 50% formamide in 2x SSC at 42 to 46 degrees C for 10 minutes (two or three times). This is a high-stringency wash that removes non-specifically hybridised probe.

Wash in 2x SSC at 42 degrees C for 5 minutes (two to three times).

Wash in 2x SSC at room temperature for 5 minutes.

Wash briefly in PBS at room temperature.

Counterstaining and Slide Mounting

Equilibrate samples briefly in PBS after the final wash.

Incubate samples in DAPI working solution for 2 to 5 minutes at room temperature in the dark. The DAPI concentration should be optimised to provide clear nuclear delineation without spectral bleed-through into probe detection channels. A starting range of 100 ng/mL to 500 ng/mL is commonly used.

Wash with PBS (two to three times, 3 to 5 minutes each) to remove excess DAPI.

Briefly rinse with distilled water or RNase-free water to remove salt deposits.

Remove excess liquid but do not allow samples to dry fully. Mount immediately in an appropriate antifade aqueous mounting medium, using a sufficient volume to fill the space between the sample and the coverslip without air bubbles.

For coverslip samples mounted onto slides: lower the mounting slide onto the coverslip slowly to avoid bubble formation. For tissue sections: apply mounting medium directly to the section and lower a clean coverslip.

Allow mounting medium to cure according to the manufacturer's guidance. Seal coverslip edges with clear nail varnish or optical adhesive. Avoid silicone sealants, which can generate autofluorescence in some filter ranges.

Store mounted slides in the dark at 4 degrees C. Image as soon as practicable. Fluorophore stability is variable; monitor signal integrity over time for prolonged storage.

Imaging Parameters and Acquisition

Image acquisition parameters must be configured for the specific fluorophores in use and the widefield microscope platform available. The following represents general guidance; final settings must be determined empirically and remain consistent across all experimental and control samples within an experiment.

Use an objective appropriate for the expected signal size and spatial resolution required. For nuclear foci (DNA FISH signals) and RNA FISH signals, a 63x or 100x oil immersion objective with a numerical aperture of 1.3 or greater is appropriate for most applications.

Acquire images in sequential channel mode (one fluorophore at a time) to minimise bleed-through between channels, which is a common source of false co-localisation signal in multi-colour FISH.

Set exposure times and gain using the positive control samples or a validated reference sample. Maintain identical acquisition settings across all samples within an experiment.

Acquire z-stack images where three-dimensional localisation of signals (particularly for DNA FISH loci) is required. z-step size should be appropriate for the depth of field of the objective in use.

Include separate acquisition of a no-probe control in every imaging session to establish the autofluorescence level in each channel.

Required Controls

A robust control strategy is essential for the interpretation of combined DNA and RNA FISH data. The controls listed below should be included in every experiment. Omission of any of these controls should be justified and documented.

Species Considerations

This protocol is applicable to mammalian cell and tissue samples. For non-mammalian species, several parameters require re-evaluation: fixation chemistry (PFA concentration and duration), denaturation temperature and formamide concentration (adjusted for genomic GC content), and the composition of blocking reagents (COT-1 DNA equivalents may need to be species-matched). Published FISH protocols for model organisms such as Drosophila, zebrafish, or plant systems use substantially modified workflows that are outside the scope of this master protocol.

FFPE versus Frozen Tissue

FFPE tissue generally requires more aggressive pre-treatment (antigen retrieval, proteinase K digestion) to achieve comparable probe penetration to frozen sections or cultured cells. RNA FISH signal intensity in FFPE is often lower than in frozen material and is strongly dependent on pre-analytical tissue handling. If quantitative RNA FISH data are required, frozen material is preferred where feasible. FFPE is acceptable for qualitative or semi-quantitative applications when RNA integrity in the block has been confirmed.

Troubleshooting

Control

Purpose

Expected Outcome

No-probe control (RNA channel)

Establishes autofluorescence baseline

No discrete fluorescent foci; diffuse or absent background only

No-probe control (DNA channel)

Establishes autofluorescence baseline

No discrete fluorescent foci

RNase A pre-treatment

Confirms RNA probe specificity

Complete loss of RNA FISH signal; DNA FISH signal preserved

DNase I pre-treatment

Confirms DNA probe specificity

Complete loss of DNA FISH signal; RNA FISH signal preserved (if performed prior to DNA FISH step)

Positive control (known expressing sample)

Confirms RNA FISH performance across experiment

Consistent signal in expected subcellular location

Known chromosomal locus control (DNA)

Confirms DNA FISH performance and denaturation efficiency

Expected copy number and locus morphology

Single-plex RNA FISH reference

Validates that combined protocol does not reduce RNA signal compared to RNA-only FISH

Comparable signal intensity and frequency

Observation

Likely Cause(s)

Recommended Action

Absent or very weak RNA FISH signal

RNA degradation; probe concentration too low; insufficient permeabilisation; excessive post-hybridisation wash stringency

Check RNase-free conditions throughout; confirm RNA integrity using RNaseAlert or equivalent; re-optimise probe concentration and wash stringency; confirm permeabilisation is adequate for sample type

Absent or very weak DNA FISH signal

Insufficient denaturation; probe concentration too low; excessive wash stringency; incomplete probe penetration

Re-optimise denaturation temperature and time; confirm probe concentration and hybridisation conditions; reduce wash stringency; ensure adequate permeabilisation

High non-specific background (RNA channel)

Insufficient pre-hybridisation; wash stringency too low; probe concentration too high; non-specific probe binding

Extend pre-hybridisation time; increase wash stringency (reduce SSC concentration or increase temperature); reduce probe concentration; include additional blocking reagents

High non-specific background (DNA channel)

Insufficient COT-1 DNA blocking; wash stringency too low; probe concentration too high; non-specific probe binding to RNA

Increase COT-1 DNA concentration in hybridisation mix; increase wash temperature; reduce probe concentration

RNA signal absent after combined DNA FISH denaturation

RNA degraded by denaturation step; formamide or high temperature conditions incompatible with RNA preservation

Reduce denaturation temperature and/or time; confirm RNA signal preservation using sequential single-plex controls; consider separate section approach if combined protocol is not feasible for the specific probe combination

Bleed-through between fluorescence channels

Spectral overlap between fluorophores; filter sets not optimally matched to fluorophores; DAPI concentration too high

Use sequentially acquired imaging; confirm filter set compatibility with chosen fluorophores; reduce DAPI concentration; use spectral unmixing if available on the platform

Poor cellular or nuclear morphology

Over-permeabilisation; excessive proteinase K digestion (FFPE); over-denaturation

Reduce detergent concentration or incubation time; optimise proteinase K conditions; reduce denaturation time or temperature

Coverslip or section lifting during protocol

Insufficient cell adhesion or tissue section adhesion

Use pre-coated coverslips (e.g., poly-L-lysine or fibronectin); use positively charged slides for tissue; reduce agitation during permeabilisation and wash steps

Products and support for this protocol

Browse FISH probes. Confirm application validation, species reactivity, sample type and detection requirements before selection.

Contact ABMIUM scientific support for product selection and troubleshooting.

For research use only. This protocol is general guidance and does not replace product-specific instructions, institutional safety procedures or local risk assessments.