Pour le prélèvement de sang total et la préservation de l'ADN et de l'ARN circulants exempts de cellules
Détails
Supporting Data
Figure 1. Effect of ambient temperature storage on cf-DNA (pDNA), exemplified by the short Alu (115bp) fragment, and genomic DNA (gDNA), exemplified by the large Alu (247 bp) fragment.
Blood samples were drawn into either: 1) EDTA tubes, 2) Competitor tubes or 3) Norgen's cf-DNA/cf-RNA Preservative Tubes and stored at room temperature. Aliquots of blood were removed at the indicated times and the plasma was separated. DNA was isolated, and pDNA and gDNA concentrations were determined by real-time PCR using a short ALU gene target (115 bp) representing the pDNA and a long ALU gene (247 bp) representing the gDNA. Levels of these two fragments should stay the same for the duration indicating stabilization and no hemolysis. As expected, there was no stabilization of cf-DNA and extensive hemolysis in the EDTA tube. Competitor showed significantly lower Ct values for both genes after 14 days, whereas cf-DNA was stable for 30 days at room temperature for Norgen's cf-DNA/cf-RNA Preservative Tubes.
Figure 2. No plasma volume loss after shipping/transportation.
Blood was drawn from 6 different donors in duplicate. One set was kept in the lab at room temperature and the other was packed in an insulated box and shipped from Thorold, ON via overnight air freight to Winnipeg, MB and then back to Thorold ON (elapsed time 72 h). Upon return, preserved samples were stored at room temperature for 7 days before plasma was separated. The plasma volume recovered from Norgen's cf-DNA/cf-RNA Preservative Tubes did not change before shipping or after shipping (6-7 mL recovered plasma). For both Competitor tubes and EDTA tubes the plasma volume recovered before shipping was ~ 4 mL and after shipping was ~ 2.5 mL.
Figure 3. Hemolysis of collected blood measured over time.
Blood samples were drawn into: 1) EDTA tubes, 2) Competitor tubes and 3) Norgen's cf-DNA/cf-RNA Preservative Tubes and stored for up to 30 days. Hemolysis was determined by measuring the absorption of free hemoglobin in plasma from 3 subjects at 414 nm over several time points. Mean absorption is shown. The amount of free hemoglobin increased rapidly with each additional storage day in the EDTA tubes and Competitor tubes, and remained relatively constant in Norgen's cf-DNA/cf-RNA Preservative Tubes.
Figure 4. Prevent cell lysis and the release of gDNA and accumulation of apoptotic ladder in plasma.
Blood samples drawn into three different tubes (Norgen's cf-DNA/cf-RNA Preservative Tubes, EDTA, and Competitor) and stored for up to 30 days. Norgen's cf-DNA/cf-RNA Preservative Tubes helps prevent the release of high molecular weight gDNA into plasma while also minimizing the accumulation of contaminating apoptotic ladder from dying peripheral blood leukocytes. As indicated in the black circle, gDNA contamination is at a very low level as compared to both the Competitor and EDTA tubes. This is indicative of very low levels of cell lysis and subsequent release of gDNA into the preserved plasma sample.
Figure 5. Effect of high temperature (37°C) storage for 8 days.
Blood samples were drawn into either EDTA tubes, Competitor tubes or Norgen's cf-DNA/cf-RNA Preservative Tubes and stored at 37°C. cf-DNA was then isolated from processed plasma. gDNA concentration was determined by real-time PCR using a long ALU gene target (247 bp). The gDNA - target in EDTA tubes showed a significant drop in the Ct. value after 1 day of storage at 37°C compared to the initial time point, and continued to drop until the 8th day of storage. For Competitor, the gDNA - target remained stable up to the 4th day of storage and then the Ct. values started to significantly drop, indicating poor stabilization beyond day 4. Norgen's cf-DNA/cf-RNA Preservative Tubes stabilized samples for 8 days.
Figure 6. High Quantity of cf-DNA from Plasma preserved using Norgen's cf-DNA/cf-RNA Preservative Tubes.
Blood samples from the same donor were drawn into either Competitor tubes or Norgen's cf-DNA/cf-RNA Preservative Tubes and stored at room temperature for 7 days. Norgen's cf-DNA/cf-RNA preservative Tube recovered 6.5mL plasma whereas the Competitor's tube recovered 3.5mL plasma. The cf-DNA was then isolated from the entire plasma volume recovered from each tube using Norgen's Plasma/Serum Cell-Free Circulating DNA Purification Midi Kit (Cat. 55600) and Norgen's Plasma/Serum Cell-Free Circulating DNA Purification Maxi Kit (Cat. 55800). The quantity of DNA recovered from both samples were assessed using the Agilent Bio-analyzer High Sensitivity DNA Chip. As can be seen in the bioanalyzer trace, Norgen's preservative tube yielded more cf-DNA (Blue peak) as compared to the cf-DNA recovered from the Competitor's preservative tube.
Figure 7. Effect of ambient temperature storage on cf-RNA, exemplified by the 18S rRNA transcript, HPRT1 mRNA transcript and miR-21.
Blood samples were drawn into either: 1) Competitor tubes or 2) Norgen's cf-DNA/cf-RNA Preservative Tubes. Competitor’s tubes were stored at room temperature for 14 days whereas Norgen’s cf-DNA/cf-RNA Preservative Tubes were stored for 30 days at room temperature. At the indicated times each tube was processed and the plasma was separated. Cf-RNA was isolated and the stability of the purified cf-RNA was determined by rt-qPCR amplification targeting the 18S rRNA transcript, HPRT1 mRNA transcript and miR-21. Levels of these three targets should stay the same for the duration indicating stabilization. Competitor showed significantly higher Ct values for the three targets after 7 days indicating cf-RNA degradation, whereas cf-RNA was stable for 30 days at room temperature for Norgen's cf-DNA/cf-RNA Preservative Tubes.
Figure 8. Effect of ambient temperature storage on plasma protein preservation for 30 days.
Blood samples were drawn into Norgen's cf-DNA/cf-RNA Preservative Tubes and were stored for 30 days at room temperature. At the indicated times each tube was processed for plasma recovery and proteins were purified from 50 µL plasma using Norgen’s ProteoSpin™ Abundant Serum Protein Depletion Kit (Cat. 17300) . The stability of the purified A) depleted – plasma proteins and B) highly abundant plasma proteins was evaluated on a 12% SDS-PAGE Gel. Both the depleted – plasma proteins and the highly abundant plasma proteins were found to be stable for 30 days at ambient temperature without any sign of protein degradation.
Données justificatives
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Spécifications du kit
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Taille du tube
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16 x 100 mm
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| Type de tube | Tube PET |
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Volume de la prise de sang
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8,4 ml dans des tubes de 10 ml
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| Volume du conservateur |
1.6 mL
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| Volume de plasma récupéré | Environ 6 à 7 ml (non affecté par l'expédition) |
| Anticoagulant |
Propriétaire
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| Conservateur |
Non formaldéhyde - Agent de conservation breveté
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Durée de conservation de l'ADN-cf |
Jusqu'à 30 jours à température ambiante (15 à 25°C), jusqu'à 8 jours à 37°C
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Durée de conservation de l'ARNcf |
Jusqu'à 30 jours à température ambiante (15 à 25°C)
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Durée de conservation des CTC |
Jusqu'à 14 jours à température ambiante (15 à 25°C)
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Conditions de stockage et stabilité du produit
- Conservés à 15 à 30°C, les tubes de conservation d’ADN-cf/ARN-cf de Norgen non remplis sont stables jusqu'à la date de péremption. Voir la date de péremption sur l'étiquette du tube.
- Les tubes de conservation d’ADN-cf/ARN-cf de Norgen non remplis peuvent être stockés/expédiés à court terme à une température comprise entre -20°C et 37°C pendant 10 jours maximum.
- Ne pas congeler les tubes de conservation d’ADN-cf/ARN-cf de Norgen non remplis pour un stockage à long terme de plus de 10 jours. La congélation de tubes non remplis peut entraîner une perte de vide et une précipitation
- L'ADN acellulaire contenu dans les échantillons de sang prélevés dans les tubes de conservation d’ADN-cf/ARN-cf de Norgen est stable jusqu'à 30 jours lorsqu'il est conservé à température ambiante (15 à 25°C)
- L'ADN acellulaire contenu dans les échantillons de sang prélevés dans les tubes de conservation d’ADN-cf/ARN-cf de Norgen est stable jusqu'à 8 jours lorsqu'il est conservé à 37ºC.
- L'ARN exempt de cellules contenu dans les échantillons de sang prélevés dans les tubes de conservation d’ADN-cf/ARN-cf de Norgen est stable jusqu'à 30 jours lorsqu'il est conservé à température ambiante (15 à 25°C).
- Les cellules tumorales circulantes (CTC) présentes dans les échantillons de sang prélevés dans les tubes de conservation d’ADN-cf/ARN-cf de Norgen sont stables jusqu'à 14 jours à température ambiante (15-25ºC).