cf-DNA/cf-RNA preservative tubes for whole blood collection

cf-DNA/cf-RNA Preservative Tubes are designed for whole blood collection and the preservation of cell-free circulating DNA and RNA.

Image Credit: Norgen Biotek Corporation

Image Credit: Norgen Biotek Corporation

Image Credit: Norgen Biotek Corporation

Image Credit: Norgen Biotek Corporation

Image Credit: Norgen Biotek Corporation

Image Credit: Norgen Biotek Corporation

Image Credit: Norgen Biotek Corporation

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Size

50 Tubes

Features and benefits

• cf-DNA and cf-RNA isolation and preservation from a single tube
• Preservative without fixative, no DNA cross-linking
• Preserve cf-DNA/ct-DNA for up to 8 days at 37 °C and 30 days at room temperature
• Preserve cf-RNA at room temperature for 30 days
• Preserve Circulating Tumor Cells (CTCs) at room temperature for 14 days
• No loss of plasma volume following shipping or transit
• Stops hemolysis to improve plasma separation
• Prevents apoptosis of blood cells and genomic DNA fragmentation
• Generate plasma of superior quality and quantity. Cf-RNA, Ct-DNA, and cf-DNA
• 8.7 mL of blood was vacuum-drawn and placed in 10 mL tubes.
• The plasma isolation process with this kit complies with ISO 20186-3:2019.

The closed, evacuated plastic tubes known as Norgen's cf-DNA/cf-RNA Preservative Tubes are used to collect and preserve circulating tumor DNA, cf-RNA, and circulating tumor cells in human whole blood samples while they are being stored and transported.

An inhibitor-free cf-DNA/ct-DNA/cf-RNA can be purified and concentrated in a very small elution volume by using these tubes in conjunction with Norgen's Plasma/Serum Cell-Free Circulating DNA Purification Kits or Norgen's Plasma/Serum RNA Purification Kits.

Any other cf-DNA and/or cf-RNA purification technique can be used with plasma recovered from Norgen's cf-DNA/cf-RNA Preservative Tubes. The purified cf-DNA, ct-DNA, and cf-RNA can be used in any downstream application assay, such as PCR, qPCR, rt-qPCR, methylation-sensitive PCR, Southern Blot analysis, gene expression analysis, microarrays, and NGS.

This product is not intended for use in diagnostic procedures but only for research purposes.

Performance

The process of collecting and processing whole blood for the subsequent purification of cf-DNA and/or cf-RNA is streamlined by Norgen's cf-DNA/cf-RNA Preservative Tubes. By preserving and inhibiting the blood's programmed cell death (apoptosis), the cf-DNA/cf-RNA Preservative Tubes stop intracellular DNA/RNA from escaping into plasma.

Samples of whole blood can be transported and kept at room temperature for the plasma processing that follows. At room temperature, the levels of cf/ct DNA and cf-RNA remain constant for up to 30 days. Additionally, the cf-DNA remains stable at 37 °C for up to 8 days.

There will be no buffy coat produced when blood collected on Norgen's cf-DNA/cf-RNA tubes is processed for plasma recovery, and the circulating tumor cells (CTCs) will be found at the tube's bottom with the blood sediment.

CTCs can be extracted from the blood sediment or whole preserved blood for cell sorting, cf/DNA purification, or cf-RNA purification during their 14-day stability period at room temperature.

Concentrated, superior, and inhibitor-free cf-DNA/ct-DNA or cf-RNA for any subsequent use is produced by plasma samples made from whole blood samples collected into Norgen's cf-DNA/cf-RNA Preservative Tubes and purified using Norgen's Plasma/Serum Cell-Free Circulating DNA Purification Kits or Norgen's Plasma/Serum RNA Purification Kits.

Workflow

Image Credit: Norgen Biotek Corporation

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Details

Supporting data

Figure 1. Effect of ambient temperature storage on cf-DNA (pDNA), exemplified by the short Alu (115 bp) 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. Image Credit: Norgen Biotek Corporation

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 hours). 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. Image Credit: Norgen Biotek Corporation

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. Image Credit: Norgen Biotek Corporation

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. Image Credit: Norgen Biotek Corporation

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 8^th day of storage. For Competitor, the gDNA - target remained stable up to the 4^th 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. Image Credit: Norgen Biotek Corporation

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 6.5 mL plasma, whereas the Competitor's tube recovered 3.5 mL 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) than the cf-DNA recovered from the Competitor's preservative tube. Image Credit: Norgen Biotek Corporation

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. Image Credit: Norgen Biotek Corporation

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. Image Credit: Norgen Biotek Corporation

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Kit specifications

Source: Norgen Biotek Corporation

Storage conditions and product stability

1. Unfilled Norgen cf-DNA/cf-RNA Preservative Tubes remain stable until the expiration date when kept at 15–30 °C. The expiration date is on the tube label.
2. Unfilled Norgen cf-DNA/cf-RNA Preservative Tubes may be warehoused or shipped for a maximum of 10 days at -20 °C to 37 °C.
3. Unfilled Norgen cf-DNA/cf-RNA Preservative Tubes should not be frozen for longer than 10 days. Unfilled tubes that are frozen may lose their vacuum and precipitate.
4. When kept at room temperature (15–25 °C), cell-free DNA in blood samples obtained in Norgen's cf-DNA/cf-RNA Preservative Tubes remains stable for up to 30 days.
5. When kept at 37 ºC, cell-free DNA in blood samples taken in Norgen's cf-DNA/cf-RNA Preservative Tubes remains stable for up to eight days.
6. When kept at room temperature (15–25 °C), cell-free RNA in blood samples obtained in Norgen's cf-DNA/cf-RNA Preservative Tubes remains stable for up to 30 days.
7. Blood samples obtained in Norgen's cf-DNA/cf-RNA Preservative Tubes contain circulating tumor cells (CTCs) that remain stable at room temperature (15–25 ºC) for up to 14 days.