ABSTRACT
BACKGROUND: Cell-free DNA (cfDNA) circulating in blood is currently used for noninvasive diagnostic and prognostic tests. Minimizing background DNA is vital for detection of low abundance cfDNA. We investigated whether a new blood collection device could reduce background levels of genomic DNA (gDNA) in plasma compared to K(3) EDTA tubes, when subjected to conditions that may occur during sample storage and shipping. METHODS: Blood samples were drawn from healthy donors into K(3) EDTA and Cell-Free DNA™ BCT (BCT). To simulate shipping, samples were shaken or left unshaken. In a shipping study, samples were shipped or not shipped. To assess temperature variations, samples were incubated at 6°C, 22°C, and 37°C. In all cases, plasma was harvested by centrifugation and total plasma DNA (pDNA) assayed by quantitative real-time polymerase chain reaction (qPCR). RESULTS: Shaking and shipping blood in K(3) EDTA tubes showed significant increases in pDNA, whereas no change was seen in BCTs. Blood in K(3) EDTA tubes incubated at 6°C, 22°C, and 37°C showed increases in pDNA while pDNA from BCTs remained stable. CONCLUSIONS: BCTs prevent increases in gDNA levels that can occur during sample storage and shipping. This new device permits low abundance DNA target detection and allows accurate cfDNA concentrations.
Subject(s)
Blood Specimen Collection/instrumentation , DNA/blood , Specimen Handling , Anticoagulants/blood , Anticoagulants/pharmacology , Blood Preservation , Cell-Free System , Edetic Acid/blood , Edetic Acid/pharmacology , Female , Humans , Male , Plasma , Temperature , TransportationABSTRACT
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ABSTRACT
OBJECTIVE: To investigate whether a new blood collection device stabilizes cell-free RNA (cfRNA) in blood post-phlebotomy when compared to collection using K(3)EDTA tubes. DESIGN AND METHODS: Blood samples were drawn from healthy donors into K(3)EDTA tubes and Cell-Free RNA BCTs (BCTs) and stored at room temperature (20-25 °C). At specified time points (days 0-3), plasma was separated and cfRNA was extracted. Reverse transcription real-time PCR was used to quantify mRNA for c-fos, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and for 18S rRNA. RESULTS: Blood drawn into K(3)EDTA tubes showed a steady increase in RNA concentration over 3 days of ex vivo incubation. Blood drawn into BCTs showed no statistically significant change in RNA copy number except for GAPDH on day 3. CONCLUSIONS: The novel chemical cocktail contained in the new device allows for the stabilization of cfRNA in blood samples at room temperature, which potentially enhances the clinical utility of cfRNA.
Subject(s)
Blood Cells/physiology , Blood Preservation/instrumentation , Blood Specimen Collection/instrumentation , RNA, Messenger/blood , Actins/genetics , Cell Survival , Edetic Acid/chemistry , Enzyme Inhibitors/chemistry , Female , Genes, fos , Glyceraldehyde-3-Phosphate Dehydrogenases/genetics , Humans , Male , RNA Stability , RNA, Ribosomal, 18S/genetics , Real-Time Polymerase Chain Reaction , Ribonucleases/antagonists & inhibitors , Ribonucleases/bloodSubject(s)
Blood Chemical Analysis/standards , Diabetes Mellitus/diagnosis , Glycated Hemoglobin/metabolism , Biomarkers/blood , Blood Chemical Analysis/instrumentation , Calibration , Diabetes Mellitus/blood , Diabetes Mellitus/therapy , Humans , Materials Testing , Predictive Value of Tests , Quality Control , Reference Standards , Reproducibility of ResultsABSTRACT
Thioltransferase (or Grx) belongs to the oxidoreductase family and is known to regulate redox homeostasis in cells. Mitochondrial Grx2 is a recent discovery, but its function is largely unknown. In this study we investigate Grx2 function by examining its potential peroxidase activity using lens epithelial cells (LEC). cDNA for human and mouse Grx2 was cloned into pET21d(+) vector and used to produce respective recombinant Grx2 for kinetic studies. cDNA for human Grx2 was transfected into human LEC and used for in vivo studies. Both human and mouse Grx2 showed glutathione (GSH)-dependent and thioredoxin reductase (TR)-dependent peroxidase activity. The catalytic efficiency of human and mouse Grx2 was lower than that of glutathione peroxidases (2.5 and 0.8x10(4) s(-1) M(-1), respectively), but comparable with TR-dependent peroxiredoxins (16.5 and 2.7x10(4) s(-1) M(-1), respectively). TR-dependent peroxidase activity increased 2-fold in the transfected cells and was completely abolished by addition of anti-Grx2 antibody (Ab). Flow cytometry (FACS) analysis and confocal microscopy revealed that cells preloaded with pure Grx2 detoxified peroxides more efficiently. Grx2 over-expression protected cells against H2O2-mediated disruption of mitochondrial transmembrane potential. These results suggest that Grx2 has a novel function as a peroxidase, accepting electrons both from GSH and TR. This unique property may play a role in protecting the mitochondria from oxidative damage.
