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 can provide 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.

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).

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).

Please refer to RNA-4RD recruitment pathway and FAQs.

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.

Estimated time-to-reporting is 4-6 months. A rapid RNA Diagnostic testing pipeline (PCR-based) 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 case. 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.

*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 uswithin 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 underconfluence and overconfluence 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 underconfluent, 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: Sophie Conroy (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

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.

• 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.