Information for Medical Researchers
Introduction
The WADB (WADB) is a National Health and Medical Research Council (NHMRC) Enabling facility which provides the infrastructure, consumables and laboratory personnel for low cost biospecimen processing and dual-site storage of human DNA samples in Western Australia for medical research purposes.
The WADB itself does not recruit these donors, but stores DNA for medical researcher's who have collected a DNA sample from consenting donors participating in ethically approved research studies. The WADB has established a common storage and management system for biospecimens in WA that meets best-practice standards.
A key aim of the WADB is to facilitate access to human DNA collections by the broader Western Australian and Australian research communities. By encouraging more medical researchers to use the WADB facilities it is anticipated that significant new initiatives and collaborations relevant to gene discovery, clinical and genetic epidemiology, new therapies and preventive medicine will ensue at the national and international level.
Samples stored by the WADB
The WADB stores DNA extracted from any human biological specimen. In 2009 the WADB introduced the handling and storage of plasma and serum. The infrastructure set up by the WADB allows for DNA storage at -40°C and serum/plasma/RNA storage at -80 °C. Storage temperatures lower than this (such as -80°C or -196°C), are not currently available at the WADB.
Biological samples the WADB will routinely extract DNA from
The WADB will extract DNA from any human biological sample however the most common sources to date are buffy coat (white blood cell layer collected from anticoagulated whole blood) and saliva (DNA in saliva comes from buccal epithelial cells and white blood cells from salivary glands). The WADB will extract RNA by arrangement. Other biological samples: If you have collected biological specimens other than blood or saliva (eg buccal swabs, bone marrow, toenail clippings, hair etc) you are advised to first contact the WADB to discuss so that arrangements can be made to purchase any necessary consumables.
Preferred sample type if collecting blood (DNA)
The preferred sample is fresh anti-coagulated whole blood (EDTA) supplied in its original collection tube (alternative anticoagulants such as acid citrate dextrose (ACD)* or Lithium Heparin** will also be suitable for DNA extraction but please note there are limitations associated with heparin). Ideally fresh blood samples should be sent to the WADB within 24 - 48 hrs of phlebotomy. If however this is not possible please see below for recommended storage conditions.
* Do not use glass collection tubes
** please note, there are reports in the literature advising heparin may have an inhibitory effect on enzyme-mediated reactions such as polymerase chain reaction (PCR). You are advised to research this deleterious effect prior to selecting the anticoagulant type for your study.
Preferred sample type if collecting saliva (DNA)
There are several saliva collection kits on the market that can be used for obtaining DNA. The WADB has extensive experience in extracting DNA from the Oragene® DNA Self collection kit (DNA Genotek, Canada), including the neonate sponges kit. It is the responsibility of the medical researcher to purchase the extraction kits themselves. If you have used a kit from any other manufacturer you are advised to notify the WADB prior to sending the specimen in for processing so arrangements can be made to purchase any necessary consumables.
Recommended storage conditions prior to transfer to WADB (DNA)
Blood (DNA/Plasma)
Within 24 - 48 hrs: Where anticoagulated blood is supplied fresh (within 24 - 48 hours) the WADB staff will centrifuge the blood and collect the buffy coat and/or plasma# from the blood sample using its Standard Operating Procedures (SOPs). If the plasma is not being retained for research purposes the fresh anticoagulated whole blood tube can be stored unspun (uncentrifuged) at 15 – 25 °C for up to 14 days## . If this ambient temperature cannot be maintained during this period the samples should be stored at +4 °C.
# Please note that the WADB cannot guarantee the plasma proteins of interest to your research will remain stable during the intervening perio between phlebotomy and storage (48 hrs). Special handling and processing arrangements should be implemented if your proteins of interest are not stable unless collected and frozen in less than 48 hours. It is therefore highly advisable that each researcher familiarises themselves with the time-lag period and conditions of collection/handling/storage in which their proteins of interest remain stable (this will vary between proteins)
## Please note any DNAse activity in plasma may reduce the overall yield of DNA from samples stored unspun (intact) for 14 days. It is therefore advisable to process the samples within24 - 48 hours where possible. Where this is not possiblethe samples should be centrifuged according to the instructions outlined below and the plasma removed. In our hands no deleterious effect has been noticed but there is evidence in the literature that suggest DNAses may degrade DNA in plasma.
3 - 14 days: The blood can be transported to the WADB in its original collection tube without centrifugation as described above unless you wish to retain the plasma layer for your study and more than 48 hours has elapsed since phlebotomy in which case you are advised to centrifuge the blood tube (approx 2450 RCF (x g) for 10 minutes at room temperature) and remove and retain the plasma layer for your study in your own laboratory (preferably in 2 ml flat bottom, screw cap tubes with O-ring – please contact the Manger of the WADB to discuss suitable options). The plasma should be stored at a minimum of -80 °C. Once the plasma is removed the blood tube should be sent to the WADB with the underlying layers (buffy coat and red blood cells) left intact. It should be stored and transported upright at +4 °C if an ambient temperature of approximately 15 – 25 °C cannot be met during the transport phase.
14 - 30 days (One month): Where blood is expected to be retained by the Custodian for between 14 -30 days the blood tube should be centrifuged (approx 2450 RCF (x g) for 10 minutes at room temperature) and the buffy coat layer transferred to a 10 ml sterile polypropylene centrifuge tube (the tube must have a conical bottom and screw cap lid) and preferably stored at -80 °C. It is acceptable, not ideal, to store the samples for short periods of time at lower temperatures but this should be a minimum of -20 °C and preferably -40 °C. Please note: the WADB can advise on a standardised protocol for obtaining the buffy coat and/or plasma if required.
> 30 days: Whole blood (anticoagulated) that is retained for periods longer than 1 month should be centrifuged at approx 2450 RCF (x g) for 10 minutes at room temperature and the buffy coat layer transferred to a 10 ml sterile polypropylene centrifuge tube (the tube must have a conical bottom and screw cap lid). Buffy coats should be stored at -80 °C especially where prolonged periods between collection and extraction will be experienced (this is to maximise the integrity of the white blood cells during storage). Storage at +4 °C is not recommended for long term storage of buffy coats. If the blood cannot be centrifuged and separated into its component parts then it is recommended the sample be frozen as whole blood at -80 °C (or where this is not possible then at a minimum of -20 – -40 °C)+.
+ The WADB can successfully extract DNA from frozen whole blood (fresh frozen or archived) but prefers to extract DNA from fresh or frozen buffy coats) If the blood samples or buffy coats have been stored for a long period of time at 'less than optimal temperatures' you are advised to contact the WADB first to discuss.
Serum
The WADB will process and store serum upon request. A plain clotted (plastic) or Serum Separator tube are both suitable for this purpose.
Within 24-48 hours: Where plain clotted blood (or serum separator tube) is supplied fresh (within 24 - 48 hours) the WADB staff will centrifuge the blood and collect the serum# from the blood sample using its Standard Operating Procedures (SOPs).
# Please note that the WADB cannot guarantee the serum proteins of interest to your research will remain stable during the intervening period between phlebotomy and storage (48 hrs). Special handling and processing arrangements should be implemented if your proteins of interest are not stable unless collected and frozen in less than 48 hours. It is therefore highly advisable that each researcher familiarises themselves with the time-lag period and conditions of collection/handling/storage in which their proteins of interest remain stable (this will vary between proteins)
>48 hours: Where plain clotted blood tube (or serum separator tube) is expected to be retained for greater than 48 hours the blood tubes should be centrifuged (approx 2450 RCF (x g) for 10 minutes at room temperature) and remove and retain the serum layer for your study in your own laboratory (preferably in 2 ml flat bottom, screw cap tubes with O-ring – please contact the Manger of the WADB to discuss suitable options). Serum should be stored at a minimum of -80 °C.
Saliva (DNA)
It is recommended you follow the instructions for storage temperature supplied by the relevant manufacturer of the saliva collection kit used - please refer to your donor collection instructions prior to obtaining a saliva specimen from a donor to ensure the storage conditions can be met before the sample is sent to the WADB for processing.
Biological samples the WADB will extract RNA from (by arrangement only)
There are a number of collection kits on the market suitable for obtaining RNA from blood and saliva. The WADB has extensive experience in extracting RNA from whole blood using the PAXgene™ Blood RNA System - this consists of (1) PAXgene™ Blood RNA tubes (PreAnalytix™, Becton Dickinson, USA) for blood collection and stabilization and (2) PAXgene™ Blood RNA Kit (Qiagen, Germany) for silica-membrane-based RNA purification in a spin-column format. It is the responsibility of the medical researcher to purchase the specialised blood tubes and the extraction kits themselves. RNA extraction may be performed by the WADB by special arrangement so you are advised to contact the WADB prior to collection of your samples.
Recommended storage conditions prior to transfer to WADB (RNA)
Blood (RNA)
Fresh anticoagulated whole blood (when collected in PAXgene™ Blood tubes) - store upright at 18–25 °C for a minimum of 2 hours after collection. After this initial incubation the tubes may then be stored for up to 3 days at 18–25 °C or up to 5 days at 2–8°C^ prior to transfer to the WADB for extraction.
PAXgene (phlebotomy and handling storage conditions after blood collection must be strictly adhered to prior to processing by the WADB for maximum integrity of the RNA).
^ PAXgene tubes may also be stored for 6 months at -20 – -70°C.
Saliva (RNA)
To date the WADB has not been requested to extract RNA from saliva., although kits are available on the market for this purpose. It is recommended you follow the instructions for storage temperature supplied by the relevant manufacturer of the saliva collection kit used - please refer to your donor collection instructions prior to obtaining a saliva specimen from a donor to ensure the storage conditions can be met before the sample is sent to the WADB for processing..
Methodologies used by the WADB
The WADB has developed a set of Standard Operating Procedures (SOPs) that are approved for use in a National Association of Testing Authorities (NATA) accredited laboratory facility. These are reviewed twice yearly or on an as-needed basis. All researchers are welcome to contact the Manager of the WADB to discuss these protocols in more detail, especially if they plan any downstream processes that may require alternative protocols.
Storage/Elution buffer for DNA
Stock DNA
The WADB routinely elutes (dissolves) the extracted DNA in 10mM Tris 1mM disodium EDTA (TE) buffer ph 8.0. If you require the stock DNA to be eluted in water, or an alternative concentration of TE, you must specify this prior to sending any blood or saliva (or other) samples for processing to the WADB.
Diluted DNA
"Working dilutions" for downstream applications (eg preparation of samples for quantitative PCR, genotyping etc) are routinely prepared using 10mM Tris 1mM disodium EDTA (TE) buffer ph 8.0 as the diluent. If you require the diluent to be either water or an alternative concentration of TE you must specify this prior to requesting the dilution be prepared by the WADB (this will usually be done by filling out a 'Custodian Access Form', available from the Manager of the WADB).
Quality Control
The WADB determines (i) the Absorbance 260nm/280nm ratio and (ii) quantifies the concentration of the DNA using a NanoDrop® ND-1000 UV-Vis Spectrophotometer (NanoDrop Technologies Inc., USA) - a visual and numerical read out of the Absorbance spectra of DNA is obtained. If the Absorbance 260nm/280nm ratio is less than 1.8 a standardised ethanol precipitation (according to Standard Operating Procedures) may improve this ratio if the sample contains (i) unwanted cellular proteins that have not been efficiently removed during the extraction process when protocols other than WADB Standard Operating Procedures have been used; (ii) when extracted with methods that use organic solvents (eg phenol) or (iii) mucins or other glycoproteins (eg when DNA is extracted from saliva). The DNA is not routinely electrophoresed in agarose gels or tested using PCR prior to storage nor quantified using fluorescence spectrophotometry (eg Picogreen). The WADB may be able to perform these tests by special arrangement but this cannot be guaranteed. You are advised to discuss this with the WADB prior to sending any biological samples for DNA extraction.
Storage conditions in a Secure Cryofacility
The WADB routinely stores DNA samples at -40°C. The ultra-cold freezers are placed in air-conditioned highly secure, locked, cryofacilities that are monitored 24 hours a day by central plant engineering for any temperature fluctuations that may signal a freezer malfunction. The secured facility can only be accessed using swipe-card or key pad technology by authorised personnel. The number of persons with the authority to enter is limited to a core group and entry and exit is monitored.
All DNA samples are split equally into two vials and stored at two geographically separated sites (to maximise their safety in the event of fire or flood). Site 1 is located at Queen Elizabeth Medical Centre, Nedlands, Perth and Site 2 is located at Royal Perth Hospital, central business district, Perth. The DNA is stored under the same standardised conditions at each site.
Volume of DNA stored
From blood
The WADB routinely stores either 0.5 ml (500 µl) or 1.0 ml (1000 µl) of DNA depending on the starting volume of the biological specimen (eg if the starting volume is 10 - 20 mls whole blood elution is in 1.0 ml TE buffer; if starting with 5 mls whole blood elution is in 0.5 ml TE buffer). DNA samples are split into two equal aliquots except where the starting volume is less than 40 µl (this is to minimise the risk of sublimation of volumes less than 20 µl per tube).
From saliva
The WADB follows the manufacturer's instructions for elution of DNA that has been extracted from saliva (depending on which kit has been used) - the elution volume used for the extraction of DNA from 4mls saliva using the Oragene® kit is currently 0.5 ml of TE buffer (to aid maximising the concentration of the DNA).
RNA
The WADB follows the manufacturer's instructions for elution of RNA that has been extracted from whole blood using the PAXgene™ Blood RNA System. The total volume of 160 ul is aliquotted equally into eight 0.5 ml vials to reduce exposure of the stock RNA to freeze-thaw cycles.
Normalised DNA samples (“Working Dilutions”)
The WADB stores three aliquots of DNA per donor - two identical aliquots from the original extracted DNA in 0.5ml tubes (for dual site banking, as described above) and a third 'normalised' working dilution stored in 96 well plate format. Where possible the normalised sample is routinely diluted to 100 ng/µl (total volume of 120 µl) or, where this is not possible, to 50 ng/μl Samples less than 50 ng/ μl may not routinely be normalised*.
* A standard ethanol precipitation of the sample (according to Standard Operating Procedures) and elution in a smaller volume of TE buffer may allow these “low concentration samples” to be prepared to our routine specifications.
Working dilutions of any concentration and volume (in addition to the normalised dilution described above) can be prepared by the WADB (by arrangement). The WADB has developed a 'Custodian Access Form' that ensures the details are captured in writing and release of DNA is authorised by the appropriate Custodian. This form is available from the Manager of the WADB.
Labelling of DNA Samples
The DNA samples are labelled with a unique identification (ID) code that is encrypted in a 2-dimensional (2D) barcode using data matrix symbology that is unreadable by the human eye. The major benefit of using 2D symbology is that a greater number of characters can be incorporated into a 2D barcode which takes up less printable space than a 1D barcode (important when printing onto small labels suitable for the DNA vials). Even when scanned using a laser scanner this unique code does not contain any information that will allow the person to be identified (eg it does not contain the person's name, nor medical record number etc).
The barcode is printed onto nylon cloth material that withstands laboratory chemicals and harsh temperatures (high and low). The labels can be applied to the vials even when they have been stored in freezers - this means the DNA samples do not necessarily have to be thawed to room temperature for labelling to occur. The unique ID code system was developed and implemented by the Western Australian Genetic Epidemiology Resource (WAGER).
If you wish to store DNA in the WADB you will be required to first contact the WADB and WAGER to discuss the labelling requirements for your study prior to sending any samples for processing. Often the original ID code can be incorporated directly into the barcode but if this is not possible (due to any number of reasons) the barcode ID will be generated automatically by the WAGER system.
Biospecimen Tracking
As the DNA samples are labelled with a 2D barcode, they are tracked using a laser barcode scanner that links directly into the WADB laboratory information management system (the "LIMS"). The scanner used by the WADB is capable of reading both 1D and 2D barcodes allowing maximum flexibility for researchers. The two Cryofacilites are equipped with a scanner (as are the WADB laboratories) which ensures samples can be tracked at all sites.
Data storage
The WADB does not store any identifying or named data with the DNA samples. The barcode data is stored on secure, network restricted servers located within a secure physical environment. Access to these servers is protected by multiple firewalls, user authentication, authorisation schemes and digital certificates. Secure data encryption is also enforced for all network data transfer. Regular data backups are performed and RAID technology is used to ensure data availability and high performance even in the event of any hard disk failure. The electronic security exceeds all relevant Australian Security Standards.
You may wish to use the WAGER facility to host any phenotypic or clinical data (if you are not hosting this data yourself). More information about how WAGER can assist you with data storage can be obtained at www.wager.org.au or by contacting Paul White, Manager Systems Development (E: paul.white@uwa.edu.au or by P: (08) 9346 1514)
Transport of DNA
The WADB can personally transport DNA within the metropolitan region of Perth if required or Custodians can collect from the WADB if preferred. The samples are transported in a foam esky either at room temperature, on wet ice (+4°C) or on dry ice (-20°C) (in tubes or plates) depending on the researcher's preference. If DNA is to be sent interstate or internationally an International Air Transport Assocation (IATA) accredited courier company will be used. The costs of transport will usually be set by the courier company and payment will need to be met by the Custodian.
Accessing DNA for research - how?
If you are using the WADB to store DNA from a human medical research study you may access your own DNA collection at any time including ongoing regular access, within the logistical capabilities of the WADB. The WADB has developed a 'Custodian Access Form' that ensures the release of DNA samples is authorised by the appropriate Custodian. This form is available from the Manager of the WADB.
If you are a medical researcher who is not currently using the facility but wish to obtain DNA samples stored in the WADB you will need to fill out an 'External Researcher DNA Access Request Form" (available for download). The WADB cannot guarantee access to the DNA samples stored in the facility as this requires the approval of the study Custodian themselves. You are strongly advised to make contact with the study Custodian to discuss your interest in obtaining access to their DNA collection for medical research purposes. The WADB can facilitate the process but cannot release the DNA without the Custodian's authorisation. Furthermore, even if the Custodian agrees in principle to collaborate or share the DNA for research purposes you must obtain human research ethics approval to undertake the study prior to obtaining the DNA. Further details can be found in the WADB Access Policy (available for download) or contact the Manager of the WADB to discuss.
Costs
As the WADB is an Enabling facility it operates under a 'cost recovery' model (these costs are calculated per sample). There are no ongoing fees for laboratory personnel time nor infrastructure and storage (or retrieval) of DNA you have deposited in the bank unless requests are beyond the logistical capabilities of the WADB. Processing costs will be reviewed from time to time in line with any increases in the cost of consumables so you are advised to contact the Manager of the WADB to discuss the costs for processing different biological specimens before proceeding.
External researchers wishing to access DNA in the facility (and approved by the Custodian) may be asked to cover the cost of transport of DNA samples when this is interstate or internationally. We would anticipate the courier company will bill the 'owner' of the DNA Collection (the "Custodian") directly and the Custodian would request reimbursement from the external researchers themselves.
Your obligations (the Researcher)
To store DNA in the WADB facility the WADB requires that medical researcher's (the "Custodian" sign a "Memorandum of Understanding" (MOU). Each Custodian will be required to (1) notify their institutional human research ethics committee of their plans to use the WADB facility and (2) provide a copy of the approval documentation and template consent form (if used) to the WADB. The WADB has obtained reciprocal recognition or formal acknowledgement from a number of HRECs within the Perth metropolitan region so this process is expected to be straightforward with these particular committees.
The MOU clearly outlines the responsibilities of both parties (the researcher and the bank). If you are interested in finding out more or wish to view the contents of the MOU please contact the Manager of the WADB to obtain a copy. A face to face meeting (or teleconference) is highly recommended to help answer any queries you may have about the manner in which the WADB operates.
Our obligations (the WADB)
The WADB is responsible for maintaining the personnel and infrastructure required for banking DNA samples to best practice standards as well as keeping the Custodian informed of any relevant changes that may impact on their DNA collection. The WADB will also inform governing institutions if any DNA Collections are to be moved interstate or overseas. These responsibilities are outlined in detail in the MOU.
Ethics clearance and Governance
The WADB project is overseen by the University of Western Australia's Human Research Ethics Committee and ultimately it must also abide by guidelines set by the National Health and Medical Research Council of Australia. The 'day to day' running of the WADB is overseen by a Management Committee. In turn the Management Committee is overseen by an Independent Advisory Committee.
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