Abstract
Biorepository processing includes nucleic acid extractions in batch mode from a large number of blood samples from many different donors. Handling such a large number of biospecimens presents the challenge of ensuring that samples are not switched or mislabeled during processing. One approach for confirming donor identity from DNA samples is the use of multiplexed fluorescent PCR for detecting Short Tandem Repeat (STR) allelic-size polymorphisms for a set of common autosomal loci. While donor identity of DNA extracted directly from blood collected in standard tubes containing anticoagulants can be easily verified by generating STR profiles, RNA from blood collected in PAXgene Blood RNA tubes (PAXgene RNA tubes) is depleted of DNA and is not amenable to STR fingerprinting for donor identity verification. We investigated the feasibility of isolating DNA directly from blood collected in PAXgene RNA tubes for use as template for STR DNA fingerprinting for blood donor identity verification. We determined that DNA extraction can be performed manually with the QIAamp DNA Blood Minikit or on the QIAxtractor instrument with minimal pre-processing protocol additions, and that DNA isolated from blood collected in PAXgene RNA tubes is of sufficient quantity and quality for successful STR fingerprint analysis. Adaptation of quality assurance methods such as the PAXgene RNA tube DNA extraction/STR fingerprinting assay described here is a good practice that ensures that biobanking collections provide scientists with high quality, donor-verified biomaterial.
Introduction
B
RNA can be readily obtained from human blood collected in PAXgene tubes that contain stabilizing agents inhibiting RNA degradation. Blood collected in this manner can be shipped or stored at room temperature for hours with no negative effects on RNA stability. RNA obtained from these tubes is depleted of DNA, and therefore, is not a suitable template for STR fingerprinting. We investigated the feasibility of isolating DNA directly from blood collected in PAXgene RNA tubes for use as template in STR analysis to verify blood donor identity. Our results show that DNA of sufficient quantity and quality for STR analysis can be readily isolated from blood collected in PAXgene RNA tubes. DNA extraction can be performed manually with the QIAGEN QIAamp DNA Blood Minikit or on the QIAxtractor instrument with minimal pre-processing protocol additions. This method offers a rapid, quality control step in confirming donor identity from blood collected in PAXgene RNA tubes, and it is amenable to moderate throughput by adaptation to an existing QIAGEN automated DNA extraction instrument.
Materials and Methods
Blood collection
Blood from 25 volunteers was collected into Acid Citrate Dextrose (ACD) tubes or PAXgene Blood RNA tubes (PreAnalytiX GmbH, Switzerland) using an IRB-approved protocol. ACD blood samples were stored at ambient temperature for ≤3 days prior to DNA extraction. PAXgene tubes were mixed by inverting eight times and stored at −80°C for at least 2 weeks.
Genomic DNA extraction
DNA was extracted from blood ACD tubes on the QIAxtractor (Qiagen, Alameda, CA) according to the manufacturer's instructions, and directly from blood collected in PAXgene RNA tubes, using two methods based upon modifications of the existing QIAGEN QIAamp Blood Minikit protocol. After thawing, a 100 μL aliquot from the PAXgene blood tubes was transferred to a microfuge tube and spun at 13,000 g for 5 min at 4°C. The resulting pellet was resuspended in 500 μL RNAse-free water, vortexed for 10 sec, and then spun at 13,000 g for 5 min at 4°C. The supernatant was removed and discarded.
For automated DNA extraction on the QIAxtractor, the pellet was suspended in 200 μL of PBS, loaded into a QIAxtractor deep-well plate, and DNA extracted according to the manufacturer's instructions. For manual extraction, the pellet was suspended in 200 μL of PBS, 20 μL of proteinase K (40 mg/mL), and 4 μL of RNase A (100 mg/mL), and 200 μL of Buffer AL was added. The lysate was incubated for 10 min at 56°C, transferred to a PAXgene Shredder spin column and centrifuged for 2 min at 14,000 g. The flow through was diluted with 200 μL of 100% ethanol, mixed gently, added to a QIAamp spin column, and spun for 1 min at 8000 g at room temperature. Further washing and elution were performed according to the manufacturer's instructions.
Short tandem repeat analysis
Short tandem repeat (STR) analysis was performed using the Coriell 6-plex fluorescent PCR assay or the PowerPlex® 18D System (Promega Corporation, Madison, WI). The Coriell 6-plex assay detects six highly polymorphic tetra-nucleotide microsatellites FES/FPS, vWA31, D22S417, D10S526, D5S592, and VWA31, and includes primers for the amelogenin gene for sex determination. The assay has a matching probability of approximately one in three million (for details, see http://ccr.coriell.org/Sections/Search/MSK.aspx?Ref=MSK&PgId=202).
STR analysis was performed using the PowerPlex® 18D System assay according to the manufacturer's recommendations. This assay detects 13 CODIS loci (7) plus amelogenin, Penta E, Penta D, D2S1338, and D19S433. The probability of identity for the PowerPlex® 18D assay is 1.36×10−28. 7
STR fragment analysis was performed on an ABI 3730 capillary sequencer using Gene Mapper (ABI) software. DNA standards were used to create custom bins for assigning the length of STR amplicons generated in the assay. The measured size of a particular allele and all alleles of the same repeat number grouped around an integer. These alleles were then binned to that integer ±1.5 bases for a tetranucleotide repeat. All other alleles of the same microsatellite family differed in size by a multiple of four bases.
Results
Approximately 250 ng of DNA was recovered by manual extraction of a 100 μL aliquot from 10 blood samples collected in PAXgene RNA tubes. Because of the necessity for higher throughput, we successfully adapted the manual extraction protocol onto the QIAGEN QIAxtractor instrument and obtained similar DNA recoveries. The DNA obtained from both the manual and automated procedures was then subjected to STR fingerprinting using the Coriell 6-plex assay. In all cases, an identical match was confirmed when the STR profiles obtained were compared to those determined from the same subject's DNA extracted from ACD whole blood.
DNA was also prepared from an additional 15 PAXgene RNA blood samples using the automated extraction protocol and used as template in the Coriell 6-plex assay and the Promega PowerPlex® 18D System. All 15 samples showed 100% concordance in the expected STR profiles generated from either the Coriell 6-plex or Promega 18D assays when compared to DNA from blood collected in ACD tubes. These results show that DNA obtained directly from PAXgene RNA tubes is of sufficient quality and quantity for accurate STR analysis using two independent STR assays.
Discussion
RNA prepared from blood collected in PAXgene RNA tubes is depleted of DNA and cannot be used for STR fingerprinting analysis for donor identity confirmation. We investigated the feasibility of isolating DNA directly from blood collected in RNA PAXgene tubes for use as a template in fluorescent PCR STR analysis. Our results show that DNA extracted manually or via automation from PAXgene RNA tubes is of sufficient quality and quantity to be suitable for use in two fluorescent-PCR based STR assays. Although Coriell processing practices have been established to minimize sample switching, this DNA extraction/fingerprinting combination has allowed us to verify matches reliably across over 4000 blood samples received at our institute. Given the consistency and robustness of this methodology, we now use this approach as a routine quality control step for all PAXgene RNA blood samples received at Coriell, and, in fact, this method has been used to successfully flag several submitted PAXgene RNA blood samples originally misidentified at the site of collection. Quality assurance methods such as our PAXgene RNA tube DNA extraction/fingerprinting assay are good biobanking practices that ensure that biospecimen collections provide scientists with high quality, donor-verified genetic materials for genetic research.
Footnotes
Acknowledgments
We thank Drs. Gregor Balaburski, Dorit Berlin, and Cristian Pérez for helpful comments on the manuscript, and Gretchen Smith and Brittany Coker for technical expertise. Detailed protocols for the Coriell 6-plex assay and the manual and automated extraction methods are available upon request.
Author Disclosure Statement
The authors have no institutional or commercial affiliations that might pose a conflict of interest regarding the publication of this study.