RNA for Rare Diseases (RNA-4RD) project

Splice variant submission form

We take referrals from clinicians across Australia. Please submit patient details and variant information (if known) to our submission portal. We review these weekly and will respond to the referring clinician with our initial assessment within the week.

Splice variant submission form

About the project

The RNA for Rare Disease (RNA-4RD) project is a national research initiative introducing ground-breaking RNA diagnostic testing into routine clinical practice across Australia.

Led by Professor Sandra Cooper, this research project hopes to pave the way for comprehensive integration of RNA diagnostics into mainstream clinical practice to improve diagnoses of families living with rare genetic diseases or inherited cancer predisposition.

It is supported by an MRFF Genomics Health Futures Mission grant.

Prof Sandra Cooper

Prof Sandra Cooper

Sandra is the Principal Investigator for RNA4RD. Head of the Genomic Medicine group. Scientific Director of KNC.
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Splicing diagnostics information

Translating technical innovations for splicing diagnostics into clinical genomics

Kids Neuroscience Centre are executing a Sydney Health Partners NHMRC MRFF research program to evaluate the clinical utility of RNA testing to show experimentally if and how a variant in the DNA disrupts splicing.

We use two methodologies to test a patient’s RNA. The first technique sequences all of the mRNA in a patient sample. We then analyse this data to look for variations in splicing of the gene of interest in age and sex matched controls. We validate the different splicing events observed in the RNA-Seq experiment by a highly-sensitive amplification-based technical platform (reverse transcription RT-PCR) or by long read RNA sequencing (Nanopore).

Splicing studies are carried out using RNA isolated from readily available biospecimens such as blood cells, skin fibroblasts, or available frozen biopsy specimens. We need to access a tissue that has appropriate expression levels of the gene of interest to get meaningful results that canprovide supporting experimental evidence to enable clinical classification of putative splice variants.

Throughout 2023, we will evaluate the diagnostic efficacy, cost-effectiveness, clinical benefits, and family impact; of diagnostic testing of 100 families with putative splice variants in a clinical setting.

Splice variant submission portal

We have created a submission portal where putative splice variants can be submitted for review. This data is securely stored on a Research Electronic Data Capture (REDCap) server to protect patient confidentiality and automatically feeds into our reports. This enables tracking of cases and provides all variant and inheritance information we need to devise the appropriate technical strategy.

The submission portal requests identifying patient information. However, you do not need to provide identifying information for confidentiality reasons, if you prefer not to. If you do not provide identifying information, we need de-identified patient identifiers (Hospital number, or a unique code for any individual providing a biospecimen), so at our end we can be certain from whom each specimen is derived (often we study a family trio).

Splice variant submission form

Variant review

Cases submitted to the portal will be reviewed at our weekly meeting. Many Mendelian disorders can predominantly affect one tissue (neuronal, renal, cardiac etc). So we carefully check the splicing pattern for this gene in all manifesting tissues, and the tissues we can get hold of easily (e.g. blood, skin, urine cells).

You will receive an email regarding our opinion on the likelihood of splicing abnormalities and suitability of different biospecimens to infer splicing outcomes to the manifesting tissue (with respect to developmental or tissue-specific expression of a given gene).

RNA-4RD ascertainment criteria

Other options for studies

If we are unable to study a case, either because it does not meet our ascertainment criteria, or because there is not sufficient expression in a clinically accessible tissue, we may be able to refer cases to other partner studies.

Timeline for results

Estimated time-to-reporting is 4-6 months. A rapid RNA Diagnostic testing pipeline (PCRbased) remains available for cases with clinical urgency (check box in the submission form). Unfortunately, primer synthesis no longer occurs in Australia and primers now take ~1 week to arrive. Fastest possible TAT is 3 weeks.

Current SOPs, are that we need to reproducibly detect every abnormal/normal splicing event with two separate primer pairs, confirmed in two experimental repeats, with Sanger sequencing of amplicons to confirm precisely the abnormal splicing event. As there can be multiple abnormal splicing outcomes, this often requires 8 bespoke primer pairs for each mcase. This follow-up and confirmation can take time but as the resultant clinical decision making is often significant (e.g. PGD) we feel this degree of analysis is essential.

Clinical impact survey

As part of the Sydney Health Partners MRFF-funded study, metrics around the clinical impact of our splicing studies must be recorded.

Once provided with a research diagnostic report, there is a short survey to record clinical impact.

Our goal is to develop standard operating procedures (SOPs), and gather clinical impact and health economic metrics, to enable this testing to transition into an accredited diagnostic service.

Clinical impact survey

Sample requirements

Whole blood

We require 2.5 ml blood collected in a PAXgene blood RNA tube (PreAnalytiX). RNA stability:

  • up to 3 days at room temperature (15–25°C)
  • up to 5 days at 2–8°C
  • indefinitely at -20°C


We require 8 ml blood in an EDTA tube. The sample needs to reach our laboratory within 48h of collection for sufficient PBMC yields. Please keep at room temperature, storage at 4°C impact PBMC yield.


For some genes, blood is not suitable, and we may recommend a urine sample and studies of mRNA from a urothelial cell culture. This test requires two urine samples per individual collected in a standard 70 ml urine specimen container. For best chances of successful culture, the sample needs to reach our laboratory within 4 hours of collection and preferably the urine will not be from the morning void. Keep the sample at room temperature, however, if the samples will take longer than 4 hours to reach the laboratory please keep at 4°C.

Skin biopsy and fibroblast culture

For some genes, blood is not suitable, and we may recommend a skin biopsy and studies of mRNA from fibroblasts. Send for normal processing via your Molecular Genetics department and ship either a live or frozen fibroblast culture. For the shipping of live fibroblasts, please fill the culture flask completely with media that has been equilibrated in a tissue culture incubator overnight to appropriately pH and oxygenate the media. A flask completely filled with media provides oxygen for respiration and protects against 'sloshing injury' to the cell monolayer. Tighten the cap and seal with parafilm.

Please ship at room temperature directly to our laboratory.

Snap frozen biopsy

  1. Fill Dewar ½ full with liquid nitrogen and fill the cryo-safe vessel (greater than 100 mL capacity metal cup) full with isopentane (2-methylbutane).
  2. Lower the cryo-safe vessel filled with isopentane into the Dewar containing the liquid nitrogen. Avoid allowing liquid nitrogen to enter the vessel filled with isopentane when lowering into liquid nitrogen Dewar. When the isopentane starts to thicken and is within the correct temperature range (-140 °C to -160 °C), a thick layer of white, frozen isopentane crystals will form at edges of the cryo-safe vessel.
  3. Stir the isopentane as the isopentane cools to maintain a uniform temperature and an open lake of liquid isopentane large enough to accommodate the cork containing the cover slip and tissue.
  4. Lower the biopsy into precooled isopentane with the cork down and tissue specimen on top. Hold cork steadily in this position for 30 seconds (using a timer) until the freezing process is complete.
  5. Carefully remove the frozen cork and specimen from the isopentane and immediately place sample on dry ice (or in cryostat, if available).
  6. Gently remove the coverslip while keeping the sample in contact with the dry ice (or inside the cold cryostat chamber). The sample must remain on the cork.
  7. Wrap the biopsy specimen in aluminium foil that has been precooled on dry ice or in the cryostat, and place the frozen aluminium foil-wrapped specimen into the labelled Poly-con container that has also been precooled on dry ice or in a suitable cryostat.
  8. Immediately transfer the Poly-con container (on dry ice) into a -80 °C freezer.
  9. Frozen biopsy specimens must be stored frozen (-80 °C) until shipped on dry ice.

Samples for gDNA sequencing

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: Jessica Mazzarolo/Jordan Conway (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

Courier delivery address

Attention: Prof Sandra Cooper / Adam Bournazos
Kids Neuroscience Centre
The Children’s Hospital at Westmead
Level 3, Kids Research Building
Loading Dock 5, Redbank Road
Westmead NSW 2145
Ph: 61 2 9845 1442 / +61 404 126 711

Note: Please advise when the shipment is dispatched and forward the tracking number to adam.bournazos@health.nsw.gov.au

Research testing and consent

Research testing

Currently, RNA splicing studies are research testing, but we are taking steps to comply with regulatory requirements for prospective accreditation. Consent for collection, testing and storage of human biospecimens for research requires completion of the research consent forms. Patients must be provided with the relevant information to allow for informed consent to be undertaken. We use previously archived RNA from other families to provide the appropriate age and gender-matched control RNA for future cases.

For more information please see section 13, subsection 6 in the Adult and Parent / Guardian Information Sheet.

Summary to assist research consenting

  • This is research testing (for now, this project hopes to obtain health services delivery metrics to translate this testing into routine diagnostics).
  • Our studies will look at the mRNA recipe for the gene thought to be at the root cause of a family's genetic condition, using blood, skin or urine cells (samples that are relatively easy to get), in an effort to show if their genetic variant actually disrupts splicing of the RNA recipe for the protein.
  • We have had a lot of success with this new technique in helping to provide a genetic diagnosis for families, but sometimes our testing is uninformative (1/40 families tested so far).
  • We will store biospecimens from the family in our secure, private Biobank. We will keep biospecimens in the Biobank indefinitely.
  • The family can request at any time to withdraw from the study and have their samples removed from the Biobank.
  • Once we have completed testing for this family, we will send a research report to the doctor(s) who sought this testing, and the doctors and genetic counsellors will explain the results to the family.



Chi Lynch-Sutherland

Email: clynch-sutherland@cmri.org.au

Phone: 0487 103 568