Processing of samples
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NATIONAL DNA REPOSITORY
December 2004 update
Standard Operating Procedures
In 2001, under the Human
Samples Collection Initiative the BRIGHT study investigators were awarded a strategic grant to perform Epstein Barr virus (EBV) immortalisation of
peripheral blood lymphocytes (PBLs) collected from all BRIGHT
sibling-pair and TDT trio samples.
Our aim was to produce a high quality DNA bank from all individuals in the BRIGHT study resource [~ 6500 samples (3500 sib-pairs and 3000 TDT trios)].This resource could then facilitate many studies into the genetic basis of essential hypertension for many years to come.
Processing of samples
For each individual, a 10ml blood sample was taken into an ACD tube, and PBLs were prepared (two samples per individual). All samples were subsequently stored in liquid nitrogen storage facilities at the Wellcome Trust Centre for Human Genetics (WTCFHG), Oxford. For this project one PBL sample per individual was transported to the European Collection of Animal and Cell Culture (ECACC) facility in Porton Down, samples were sent in batches at regular intervals.
At ECACC, Epstein Barr virus (EBV) transformation was performed and growing cell lines were sent to London in weekly batches of 35. The samples were cultured for a further two weeks to increase the confluency of cells and were then harvested. DNA extraction was carried out on the cell pellets, and quantification using picogreen and DNA fingerprinting was performed. DNA fingerprinting is important to ensure that there has been no errors in labelling or in the transportation process.
All DNA samples will subsequently be made into master plates (50ng/ul) for use by investigators. At ECACC, four vials of transformed lymphocytes per individual will be kept and we have agreed that they will be stored in liquid nitrogen for a minimum of 5 years.
December 2004 update:
We have now performed EBV immortalisation of 3450 PBL samples at ECACC
in Porton Down. In late 2002, half of our frozen PBLs were accidently destroyed, this has reduced the resource considerably.
The overall success rate of transformation of BRIGHT samples was 85%.
We have successfully extracted DNA from 80% of the transformed samples at this time. Quantification and fingerprinting using 5 unlinked microsatellite markers is currently being done.
Average DNA yield is ~470ng/ul.
Once processing of all the samples is complete, stock
plates will be prepared. An aliquot will be sent to MRC geneservice, remaining stocks will be held by the BRIGHT PIs.
The resource when completed will be of great value for molecular genetic research into hypertension not only by BRIGHT investigators but also for other interested groups.
We have established a steering group (under a neutral
chair) that will review requests for access to the DNA Repository
and phenotypic data when it becomes available.
Standard Operating Procedures: