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Spatial Biology Services

RNA In Situ Hybridization Services

Localize target RNA expression in tissues and cells, with study-specific assay optimization, imaging, and quantitative analysis.

Discuss Your RNA ISH Study

We help research teams determine where target RNA is expressed, which cells express it, and how expression varies across tissue regions or study groups. Each project begins with a review of your targets, samples, and research objectives to define the assay approach and analysis plan.

RNA ISH for spatial gene expression

RNA in situ hybridization (RNA ISH) detects RNA inside intact cells and tissue sections, and combines spatial imaging with quantitative or semi-quantitative readouts, allowing target RNA expression to be evaluated within its tissue context. Bulk methods such as qPCR and RNA-seq report how much of a transcript a sample contains, but average across every cell in the extract; RNA ISH shows which cells carry the transcript, whether expression is uniform or restricted to a small subpopulation, and how those cells sit next to their neighbors.

Our studies are organized around the questions research teams most often need answered:

  • Target expression localization: in which cell types and tissue compartments is the RNA present?
  • Proportion of expressing cells: what fraction of cells in a region carry the target?
  • Regional and group differences: how does expression vary between tissue regions, treatment groups or time points?
  • Co-expression analysis: are two or more targets found in the same cells?
RNA ISH answers a spatial question: not simply whether a gene is detected, but where, in which cells, and at what relative level.
close-up fluorescence microscopy image of a tissue section, blue stained cell nuclei with small bright green and red punctate dots inside the cell outlines, dark background, crisp focus, scientific il

Research applications and biomarkers

RNA ISH can be applied to a wide range of RNA targets, provided a sufficiently unique target sequence can be identified and the sample RNA is of adequate quality. Each target is reviewed for sequence suitability and sample feasibility before the study begins. The approach is especially useful where protein detection is weak or impossible.

  • mRNA expression

    Map cell-type markers, receptors, cytokines and low-abundance transcripts across tissue, and separate expressing from non-expressing cells in the same field.

  • Non-coding RNA

    Localize long non-coding RNAs, which have no protein to stain, and see whether they accumulate in the nucleus or cytoplasm.

  • Viral RNA

    Identify infected cells, distinguish genomic from messenger viral RNA when suitable probes are available, and relate infection to host-cell response.

  • Microbial targets

    Detect bacterial and other microbial RNA in tissue, for example to locate organisms relative to host cells and inflammation.

  • Engineered-cell markers

    Confirm that transgenes, reporters, CAR constructs or edited sequences are expressed, and in which cells, after delivery or engraftment.

  • Antibody-limited biomarkers

    Secreted proteins, small peptides, new targets and poorly validated antibody targets can often be followed by their transcript instead, with the caveat that RNA is not a direct readout of protein.

A note on RNA versus protein

An RNA signal reflects the presence and distribution of the target RNA. It does not show protein abundance or function, and a standard RNA ISH result cannot be equated with real-time transcriptional activity. When a conclusion depends on the protein, RNA detection can be paired with protein detection in the same section where feasible (RNA-protein co-detection) rather than inferring one from the other.

What our RNA ISH service covers

Each project is scoped around your target, sample and research objective. A typical study can include the stages below.

  • Sample assessment

    Review of species, tissue, fixation history, block age and section quality to judge whether the RNA in your material is likely to support interpretable results.

  • Assay optimization

    A small pilot to tune tissue pretreatment and confirm target and control performance before proceeding with the full sample set.

  • Staining

    RNA ISH staining of study slides alongside positive and negative controls, using the approach selected during feasibility assessment.

  • Imaging

    Brightfield or fluorescence imaging with acquisition settings defined from the controls and applied consistently across the slides being compared.

  • Image analysis

    Cell segmentation and signal counting or scoring with documented, fixed criteria, producing per-cell or per-area readouts suited to the question.

  • Reporting

    A written report describing methods, quality-control findings, results and the limitations that matter for interpreting them.

Your proposal will specify the assay method, sample requirements, deliverables, timeline, and any work performed by external laboratories.

tidy laboratory bench scene viewed from above showing glass microscope slides in a staining rack, a small tray of paraffin tissue blocks, and a flask of buffer, soft daylight, shallow depth of field,
Illustrative image.

Sample requirements and compatibility

RNA ISH methods are widely used because they work on the sample types real studies depend on. Commonly supported preparations include the following. The tissues, species and fixation conditions supported for your project are confirmed during feasibility assessment.

  • FFPE tissue sections. The most widely used format, covering archival clinical material and standard animal studies.
  • Fresh-frozen sections. Often chosen when RNA integrity is the top priority or the antibody in a co-detection panel prefers unfixed antigen.
  • Fixed-frozen sections. Common for brain and other delicate tissues, where fixation precedes cryoprotection and sectioning.
  • Cultured cells and cytospins. Useful for assay development and for checking probe performance before moving to tissue.

Practical notes

FFPE: fixation time matters. Under-fixed tissue can lose RNA and over-fixed tissue can resist probe access, so the pretreatment steps may need adjusting. Manufacturer guidance for FFPE generally specifies fresh neutral buffered formalin for a defined fixation window; the relevant technical note for the method used gives the current figures. Fresh-frozen: keep sections cold until fixation on the slide, and expect morphology to be less crisp than FFPE.

Assay validation: for any sample type, validation includes positive and negative control probes on the same tissue before targets of interest are interpreted.

Information to share with us

  • Target gene or RNA, and the species it should be detected in.
  • Tissue or cell type, and the preparation (FFPE, fresh-frozen, fixed-frozen, cells).
  • Fixation type and duration, block age, section thickness and storage conditions.
  • Number of samples, groups and the comparisons you need to make.
  • The research objective and the readout you expect, such as localization, percentage of positive cells or co-expression.

Validate before you scale

Every new tissue type, species, decalcification step or fixation protocol benefits from a short pilot with positive and negative control probes. A pilot uses a few slides and can prevent a whole cohort being run on RNA that was already degraded.

Project workflow and quality control

Studies move through four stages so that decisions about target, sample and method are made on evidence before the full experiment is run.

  1. Feasibility assessment

    We review your target, species, sample type and objective, check target sequence suitability and sample quality risks, and recommend an approach.

  2. Small-scale optimization

    A pilot on representative material, with positive and negative controls, confirms that the assay performs and fixes the conditions to be used.

  3. Full study

    Study slides are stained and imaged under the locked conditions, with controls processed alongside the samples being compared.

  4. Analysis and reporting

    Images are analyzed with predefined criteria and results are documented with methods, quality-control findings and limitations.

Timelines depend on the target, the number of samples and the readout, and are set out in the project proposal.

Quality controls and study consistency

Each study is designed with appropriate positive and negative controls. Sample suitability, assay performance, imaging settings, and analysis criteria are assessed before full study execution. Reports document the methods, quality-control findings, and limitations relevant to interpretation.

Positive and negative controls

An RNA ISH run is hard to interpret without controls on the same tissue, processed in the same run.

  • Positive control probes target housekeeping genes and help assess RNA preservation and assay performance. Controls with expression levels appropriate to the tissue and assay help determine whether low-abundance targets can be interpreted reliably.
  • Negative control probes target sequences expected to be absent from the sample and help assess nonspecific signal. Unexpected staining or fluorescence is investigated before target results are interpreted.
  • Tissue-appropriate targets. Where possible, a tissue known to express the target and one known not to are included.

Standard pretreatment is a good starting point, but tissues differ in fixation history, density and protease sensitivity. Pretreatment conditions are adjusted to balance signal detection and tissue morphology, the control panel is repeated after each change, and the conditions are locked before the full study begins. Results are interpreted only when the relevant controls meet predefined acceptance criteria.

Imaging and analysis

Imaging parameters are established using appropriate controls to assess background and avoid signal saturation. Consistent acquisition settings are applied across samples intended for comparison.

Signal is counted or scored with a documented analysis method chosen for the specific assay, the signal characteristics and the research objective. No single scoring table applies to every RNA ISH method, so assay-specific guidance is not transferred between methods. Analysis is applied blind to condition where possible, and the method is reported alongside the result. For larger studies, software segments nuclei, assigns signal to cells and reports counts per cell or per area; segmentation is reviewed across representative tissue regions and signal levels, and parameters are kept fixed across slides rather than tuned separately for each image.

clean flat infographic on a white background titled From dots to data, showing a left-to-right pipeline of four panels joined by arrows: panel 1 labelled Image with a small microscope field of cells w
Schematic of a generic image-analysis pipeline. A fixed, documented analysis pipeline matters more than which software runs it.

Reading punctate signal with care

In a well-optimized assay on suitable tissue, discrete punctate signals can often be resolved, and counts per cell can serve as a proxy for relative RNA abundance. Whether a given dot corresponds to a single RNA molecule depends on the method, section thickness, magnification, probe design and the controls run alongside, so it is verified for the assay in use rather than assumed. For highly expressed transcripts, signals overlap into clusters that cannot be separated, and counts then underestimate true abundance. Weak diffuse haze across the section is usually background, not expression.

Common pitfalls we plan for

  • Autofluorescence. Aged or pigment-rich tissues can fluoresce across channels and be mistaken for signal; comparison with the negative control helps separate the two.
  • Saturated images. Saturation merges neighboring signals and distorts counts.
  • Over-digestion or drying. Excess protease damages morphology and drying causes patchy background; both undermine quantification.
  • Segmentation errors. Overlapping nuclei and thick sections lead to signal being assigned to the wrong cell.

Localization versus quantitative claims

RNA ISH is especially strong at qualitative and spatial conclusions: this cell type expresses the target, this region does not, these two transcripts are found in the same cells. It supports relative comparisons well when sections are processed together under the same settings. It is a weaker tool for absolute copy-number claims across different runs, tissues or fixation histories, and for such claims an orthogonal method is a sensible companion.

Reading the control result

A good run shows a clear positive control signal in the expected cell types and a clean negative control. Results are interpreted only when the relevant controls meet predefined acceptance criteria. The scope of controls applied to each project is defined in the proposal.

Study deliverables

Depending on the agreed scope, deliverables may include:

  • Digital images of the agreed study sections or regions of interest.
  • Positive and negative control results, with relevant quality-control observations.
  • Quantitative outputs such as signal counts, positive-cell percentages, or region-level measurements.
  • A methods summary describing the assay, imaging, and analysis approach.
  • A study report presenting findings and limitations relevant to interpretation.

Whole-slide scans, raw image files and cell-level data are provided only when they are part of the agreed scope.

Selecting an RNA ISH Approach

Assay selection depends on the target sequence, sample preservation, number of targets, and intended readout. We review these factors during feasibility assessment and specify the proposed method in the study proposal.

Probe design and signal detection

Target-specific probes hybridize to complementary RNA sequences in the sample. Depending on the assay, signals are detected directly or through amplification. Probe specificity, tissue pretreatment and background are evaluated with appropriate controls before study results are interpreted.

Readout options

Chromogenic detection
Colored signals are evaluated by brightfield microscopy alongside tissue morphology.
Fluorescent detection
Distinct fluorescence channels can support detection of multiple targets and assessment of their spatial relationships.
RNA and protein co-detection
RNA ISH may be combined with protein staining when sample preparation and assay conditions are compatible. Feasibility is assessed before a combined study is proposed.

From sample to readout

A typical workflow includes sample preparation, tissue pretreatment, probe hybridization, signal detection, imaging and analysis. Conditions are selected for the sample and assay rather than transferred unchanged between methods. Your proposal defines the method, controls and outputs for the study.

RNA ISH service FAQ

What samples can you assess for RNA ISH?

Commonly supported preparations include FFPE tissue sections, fresh-frozen sections, fixed-frozen sections, and cultured cells or cytospins. The tissues, species and fixation conditions supported for your project are confirmed during feasibility assessment.

What information do you need to scope a project?

Share your target, species, sample preparation, approximate sample count, and research objective. Fixation details, block age and the comparisons you need to make also help us assess target sequence suitability, sample quality risks and an appropriate study approach.

Can archived FFPE samples be evaluated?

Often, yes. FFPE sections are the most common sample type for RNA ISH, but results depend on fixation history, block age and RNA preservation. Control probes are run on the same tissue to help assess RNA preservation and assay performance, and pretreatment conditions are adjusted to balance signal detection and tissue morphology.

What controls are included?

Studies are designed with positive and negative controls processed on the same tissue and in the same run. Results are interpreted only when the relevant controls meet predefined acceptance criteria. The controls applied to each project are defined in the study proposal.

What data and images will I receive?

Depending on the agreed scope, deliverables may include digital images of the agreed sections or regions of interest, positive and negative control results with quality-control observations, quantitative outputs such as signal counts or positive-cell percentages, a methods summary, and a study report presenting findings and limitations.

How are project timelines determined?

Timelines depend on the target, the number of samples, the need for optimization and the intended readout. Staining of a manual assay run typically takes one to two working days, with imaging, analysis and reporting on top. The proposal sets out the timeline for your study.

Plan Your RNA ISH Study

Share your target, species, sample preparation, approximate sample count, and research objective. Our team will assess feasibility and recommend a study approach, control strategy, and analysis plan suited to your project.

Discuss Your RNA ISH Study

For research use only. Not for use in diagnostic procedures.

References and resources

  1. Femino AM, Fay FS, Fogarty K, Singer RH. Visualization of single RNA transcripts in situ. Science. 1998;280(5363):585-590. doi.org/10.1126/science.280.5363.585
  2. Raj A, van den Bogaard P, Rifkin SA, van Oudenaarden A, Tyagi S. Imaging individual mRNA molecules using multiple singly labeled probes. Nat Methods. 2008;5(10):877-879. doi.org/10.1038/nmeth.1253

For research use only. Not for use in diagnostic procedures.