Many patients remain without a molecular diagnosis despite extensive DNA-based testing. In some cases, the underlying variant affects how RNA is processed rather than altering the protein-coding sequence directly.

RNA-4RD supports individuals with suspected monogenic disorders where RNA testing may help establish a diagnosis or clarify the significance of a genetic variant.

Primary ascertainment: most common referrals for RNA testing

Context 1: Variant identified, functional evidence required

A rare variant has been identified through genomic testing and additional RNA evidence is required to:

• assess whether the variant alters RNA splicing or expression
• support variant classification
• clarify the molecular mechanism underlying disease.

Context 2: Strong clinical suspicion, genomic testing is non-diagnostic

The affected individual's phenotype strongly suggests a specific genetic disorder or limited set of genes, but previous genomic testing has been negative, incomplete or non-diagnostic. This pathway involves genome sequencing with targeted gene curation, followed by RNA studies when indicated.

Secondary ascertainment: other situations where RNA testing may be considered

RNA testing may also provide clinically useful information by helping to:

• evaluate RNA impact of variants in newly identified disease genes
• incomplete determine whether variants are in cis or trans (phasing)
• assess allele-specific expression: evaluate imprinting disorders or X-chromosome inactivation
• investigate nonsense-mediated decay
• assess selected RNA biomarkers or complex cases following multidisciplinary review.

Non-suitable cases

RNA-4RD is unlikely to be informative where:

• the clinical presentation is not consistent with a monogenic disorder
• the variant has already been classified as benign, likely benign or pathogenic without convincing contradictory evidence
• there is no plausible mechanism by which the variant could affect RNA biology
• RNA testing is unlikely to influence variant interpretation or clinical management
• the phenotype is inconsistent with the implicated gene.

If you are uncertain whether a case is suitable, we encourage submission for preliminary review by the RNA-4RD team. All cases are assessed using the RNA-4RD national ascertainment and triage framework.

Learn more on the RNA-4RD Phase II Ascertainment Criteria and Case Triage Framework.

RNA-4RD supports individuals with suspected monogenic disorders where RNA testing may help establish a diagnosis or clarify the significance of a genetic variant.

Learn more on the RNA-4RD Phase II Ascertainment Criteria and Case Triage Framework.

Cases submitted through the portal are reviewed by the RNA-4RD team at our weekly multidisciplinary meeting.

Many Mendelian disorders primarily affect specific tissues, such as the brain, kidney, heart, or muscle. As part of our assessment, we evaluate the expression and splicing profile of the gene of interest across disease-relevant tissues and compare this with accessible biospecimens such as blood, skin, or urinary cells.

Following review, you will receive an email detailing our assessment of the predicted likelihood of RNA-level impact, and the suitability of clinically accessible biospecimens for investigating RNA effects. This assessment takes into account tissue-specific and developmental gene expression patterns, as well as the relevance of the sampled tissue to the clinical phenotype.

If a case is not suitable for RNA-4RD because:

• it does not meet our ascertainment criteria or 
• the target gene is not expressed at sufficient levels in clinically accessible tissues, 

we may be able to refer the case to one of our partner research programs.

For genes with limited or absent expression in accessible biospecimens, we may recommend cross-referral to the MRFF PERSYST program. PERSYST applies gene transactivation and transdifferentiation approaches to increase expression of the target gene in patient-derived cells, enabling diagnostically informative RNA studies that would otherwise not be feasible.

In some cases, RNA testing may identify a variant-associated RNA abnormality that is consistent with pathogenicity, but uncertainty remains regarding the downstream impact on protein abundance, function, or the affected biological pathway. In these situations, we may recommend cross-referral to the MRFF RDMassSpec (Rare Disease Mass Spectrometry) program for complementary proteomic analyses to further investigate the molecular consequences of the variant.

RNA-4RD aims to provide a report within 6 months of sample receipt. This timeframe is achievable for most cases, although complex RNA findings may require additional investigations and extend reporting times.

Urgent cases

A rapid RNA diagnostic pathway is available for clinically urgent referrals. Clinicians can indicate urgency in the submission form.

Rapid testing is typically RT-PCR based, and preliminary findings can sometimes be communicated to the referring clinical team within approximately 3 weeks of sample receipt. Timeframes may vary depending on sample quality and assay requirements.

Why can some cases take longer?

RNA effects are often complex and may involve multiple abnormal transcripts or splicing outcomes. To ensure diagnostic confidence, findings typically require confirmation using independent assays and primer sets.

In some cases, additional rounds of primer design, RT-PCR, sequencing, or complementary RNA studies are needed to fully characterise the molecular effect of a variant. These extra analyses can extend turnaround times but are often essential for accurate interpretation and reporting.

*Must reach the receiving laboratory within 72 hours for processing.

Collection Instructions:

• 2.5 ml whole blood collected in a PAXgene blood RNA tube (PreAnalytiX).
• After Blood Collection gently invert the PAXgene Blood RNA Tube 8 to 10 times.
• RNA is stable at room temperature, refrigerated or frozen for the following timeframes:
  • up to 3 days at room temperature (15–25°C)
  • up to 5 days at 2–8°C
  • at least 1 year at -20°C

*DO NOT put on ice, in fridge or freezer.

**Samples must be processed by RNA testing lab within 48 hours of collection.

Collection Instructions:

• 8 ml whole blood collected into a:
  • EDTA tube, or
  • lithium heparin tube (no gel).  Please note: Use of Li-Hep tubes with no gel is essential.
• The sample must reach us within 48h of collection and be shipped and stored at room temperature. Please note: The sample is non-viable with storage at 4°C, or after 48 hours.

*DO NOT embed in ice or freeze. Place with a cool block in summer.
**Samples must be processed by RNA testing lab within 4-6 hours of collection.

Collection Instructions:

• 2 x 70 ml standard urine specimen containers per individual.
  • Please Note: Not the morning void.
• The sample must reach us within 4 – 6 h of collection and be shipped at room temperature, preferably with a cool block during hot summer months but NOT embedded in ice.
  • Please note: Cells are non-viable after 6-8 hours.

Fibroblast culture

*NO ice, fridge or freezing. DO NOT fix. 

**Biopsy must be processed by diagnostic lab within 4-6 hours.

Collection Instructions:

• Collect a 3 mm or 2 mm diameter, full-thickness skin biopsy sample, obtained under strict sterile conditions.
• Place into a sterile cryotube or specimen container with a small amount of normal saline to prevent the specimen from drying out during transport to the Diagnostic laboratory responsible for fibroblast explant culture.
• The sample must be transported at room temperature and reach the Diagnostic Laboratory within 4 – 6 h of collection for fibroblasts explant culture using standard diagnostic protocols.
  • Please note: No ice. No fridge. No freezer. No fixative.

For RNA extraction

*Sample must be processed by RNA testing lab within 48 hours.

Collection Instructions:

• Collect a 3 mm or 2 mm diameter, full-thickness skin biopsy sample, obtained under strict sterile conditions.
• Place into a 1.5 ml or 2.0 ml sterile cryotube containing RNA-later (Cooper team will provide this).
• Transport the sample at room temperature or with a Cool Block to the RNA Testing centre.

*Samples must be processed by RNA testing lab within 48 hours of collection.

Collection Instructions:

• Transfer a piece of biopsy or autopsy tissue into a sterile cryotube containing RNA-later (Cooper team will provide this).
• The sample may be transported at room temperature or with a Cool Block or On Ice to the RNA Testing centre.

*Samples must be processed by RNA testing lab within 48 hours of collection.

Collection Instructions:

• Transfer a piece of biopsy or autopsy tissue into a sterile cryotube containing RNA-later (Cooper team will provide this).
• The sample may be transported at room temperature or with a Cool Block or On Ice to the RNA Testing centre.

*Samples must be processed by RNA testing lab within 48 hours of collection.

Collection Instructions:

• The day prior to shipment, trypsinise the cells and seed at approximately 60% - 75% confluence into T25 flask (screw down cap, rather than filter cap, if possible).
  • GOAL for cell shipment:  Cells very near confluence, or an unpacked, confluent monolayer. Both under-confluence and over-confluence can limit cell viability with transit.
• Additionally on the day prior to shipment, fill a second T25 flask without any cells to near the top with culture media.  Stand this T25 upright in the tissue culture incubator overnight to pre-warm, pH balance, and oxygenate the media.  If screw cap, ensure the cap is set open to allow gas exchange.
• The day of shipment, check cell confluency. If under-confluent, defer shipment for 24 hours. If over-confluent (densely packed monolayer), trypsinise again and defer shipment for the following day.
• Stand the flask of cells to be shipped upright.  Use a 10 ml pipette to gently transfer the pre-equilibrated media to completely fill the T25 flask. Tighten the screw cap to prevent further gas exchange and seal with parafilm.  If filter cap – ensure the parafilm creates a good seal.
  • PURPOSE of filled flask: Minimise “sloshing injury” to the cell monolayer during transit.
• Ship the cells at room temperature. Transit temperature must not exceed 37^oC. Ship with a cool block if daily temperature is expected to be >37^oC. Cell viability will reduce exponentially with transit times longer than 48 hours.

*Sample must be processed by RNA testing lab within 48 hours.

Collection Instructions:

• Transfer appropriate volume (T25 – T75) of cell culture into a 15 ml tube.
• Centrifuge at room temperature at 500 x g for five minutes in a swinging bucket rotor to pellet cells.
• Remove supernatant and resuspend in 1ml of growth media and transfer to 1.5 ml tube.
• Centrifuge cells at 4°C at 500 x g for five minutes in a fixed angle rotor microcentrifuge and remove supernatant.
• Place on dry ice and incubate for five minutes to snap-freeze or resuspend in 5-10 volumes of RNAlater.

We are carrying out low pass DNA sequencing in conjunction with RNA testing for 20 context 2 cases. DNA or blood specimen sent to DNA address:

Attention: Milena Babic (RNA4RD DNA studies) 
GMP Service lab
SA Pathology, Frome Rd
Adelaide, SA 5000
Ph: (08) 8222 3648

This may be; an existing stored DNA sample, OR an additional 1-2mls of blood in an EDTA tube for fresh DNA isolation, OR a self-swab saliva sample for fresh DNA isolation

RNA Testing is currently a research test.

Although RNA testing performed through the RNA-4RD program will typically occur within accredited diagnostic laboratories, it is currently provided as a research investigation rather than a routine clinical test. RNA-4RD is helping build the evidence needed to integrate RNA testing into routine clinical care.

All RNA testing is conducted under the nationally approved Answers for Rare Diseases ethics protocol.

RNA-4RD is a defined sub-study of the broader Answers for Rare Diseases research protocol. This framework enables the RNA-4RD team to undertake RNA analyses and, where appropriate, pursue additional functional genomics investigations to help resolve complex or unsolved cases.

Depending on the findings, further studies may be considered, including:

• gene transactivation or transdifferentiation approaches
• quantitative proteomics and mass spectrometry
• enzyme activity assays
• other recognised functional studies relevant to the disease mechanism.

Participation in RNA-4RD requires informed consent. Before any RNA testing can proceed, participants (or their parent/guardian) must provide consent using the current approved RNA-4RD Participant Information and Consent Form.

This consent process enables the use of samples and data for RNA-based investigations and, where appropriate, consideration of complementary functional studies that may assist in establishing a molecular diagnosis.

The Answers for Rare Diseases Study Summary provides a simplified, illustrated overview of the study and explains the key information families should understand before consenting.

Key points for participant families:

• RNA testing is currently a research test. RNA-4RD is helping build the evidence needed to integrate RNA testing into routine clinical care.
• By analysing RNA from accessible samples, we can determine whether a DNA variant disrupts splicing or gene expression, helping to clarify its role in disease.
• RNA-4RD has supported a molecular diagnosis in approximately 65% of participating families.
• Not every RNA test will provide an answer. In some cases, RNA testing may be uninformative or may not provide sufficient evidence to support a diagnosis.
• RNA differs from DNA. RNA expression and splicing patterns vary between tissues and can change over time. Accurate interpretation relies on comparison with carefully matched reference datasets.
• RNA-4RD uses de-identified RNA data from previously consented participants as diagnostic reference datasets to improve the accuracy and interpretation of RNA test results.
• Samples and data may be retained indefinitely. Families may request removal of their stored specimens from the biobank at any time. However, de-identified RNA data that have already been incorporated into diagnostic reference datasets or used in completed analyses generally cannot be removed retrospectively.

This summary is intended to support the consent process but does not replace the full Participant Information and Consent Form.