Evaluation of Storage Tubes for Combined Analysis of Circulating Nucleic Acids in Liquid Biopsies.
Author(s): Ward Gahlawat A, Lenhardt J, Witte T, Keitel D, Kaufhold A, Maass KK, Pajtler KW, Sohn C, Schott S
Publication: Int J Mol Sci, 2019, Vol. 20, Page
PubMed ID: 30736351 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of storing blood in EDTA tubes and stabilizing tubes (Norgen, PAXgene, Streck, and Roche) on microRNA (miRNA, miR) levels in plasma and the effects of storing blood in stabilizing tubes on levels of cell-free DNA (cfDNA) in plasma and hemolysis.
Conclusion of Paper
Total plasma miRNA levels were unaffected by storage of blood in EDTA tubes for up to 18 h at 4˚C or in Streck, Roche, or Norgen tubes for up to 7 days but increased when blood was stored at room temperature for 18 h in EDTA tubes or for 24 h in PAXgene tubes. The highest miRNA yields as determined by flouormetry were also found when blood was collected in PAXgene tubes. Levels of the hemolysis marker, miR-451, were unaffected by storage of blood for 7 days in PAXgene or Norgen tubes but increased when blood was stored in EDTA tubes at 4˚C for 12 h or more and when blood was stored in Streck tubes for 5 or 7 days (P<0.001, both) or Roche tubes for 7 days (P<0.001). Further spectrophotometry showed increasing hemolysis with storage in specimens stored in Roche or Streck tubes. Importantly, the breast cancer markers miR-148b and miR-652 in some specimens were significantly affected by storage in EDTA tubes at room temperature for 18 h.
cfDNA levels were unaffected by room temperature storage of blood in Streck, Roche, Norgen, or PAXgene tubes for up to 1 week before centrifugation. DNA yields were highest when blood was collected in Norgen tubes, but the DNA peak size was smaller for these specimens (150 bp) compared to specimens collected in PAXgene, Streck, and Roche tubes (168-180 bp). While the number of copies of PIK3CA was comparable between tube types, the counts of TP53 were lower when specimens were collected in Norgen tubes than the other tube types.
Studies
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Study Purpose
This study investigated the effects of storing EDTA blood for up to 18 h at room temperature or 4˚C on miRNA levels in plasma. Blood was collected from six healthy patients into EDTA monovette tubes, inverted 10 times, and stored upright at room temperature or 4˚C for 4, 12, and 18 h. Plasma was obtained by centrifugation of blood at 1300 x g for 20 min followed by centrifugation of the plasma supernatant at 12000 x g for 10 min. Plasma was stored at -80˚C. miRNA was extracted using the NucleoSpin miRNA plasma kit and quantified using the fluorometry-based Qubit microRNA Assay Kit. Individual miRNAs were quantified using the Taqman real-time PCR. Levels of miRNA were normalized to specimens of the same tube type stored for 4 h.
Summary of Findings:
Total plasma miRNA levels were unaffected by storage of blood in EDTA tubes for up to 18 h at 4˚C, but total miRNA levels were higher in 3 of 6 specimens when blood was stored at room temperature for 18 h than when plasma was isolated immediately. Levels of the hemolysis markers, miR-451 and miR-16, were slightly higher when blood was stored at 4˚C for 12 and 18 h before centrifugation rather than processed after 4 h, but only miR-16 was increased after 18 h when stored at room temperature. The breast cancer markers miR-148b and miR-652 were significantly higher in 3 of 6 specimens stored at room temperature for 18 h than those stored for 4 h (P<0.01, both), but levels of miR-376c and miR-200c were unaffected by storage at either temperature.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform RNA Real-time qRT-PCR RNA Fluorometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage temperature 4˚C
Room temperature
Real-time qRT-PCR Specific Targeted nucleic acid miR 451a
miR-200c
miR-376c
miR-16
miR-148b
miR-652
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
Storage Storage duration 4 h
12 h
18 h
-
Study Purpose
This study investigated the effects of storing blood in Streck, Roche, PAXgene, and Norgen tubes for up to 7 days at room temperature on miRNA and cfDNA levels in plasma. Blood was collected from eight healthy patients into Streck, Roche, PAXgene, and Norgen tubes, inverted 8-10 times, and stored upright at room temperature for 4 h, 24h, 5 days, or 7 days. Plasma was obtained by centrifugation of blood at 1300 x g for 20 min followed by centrifugation of the plasma supernatant at 12000 x g for 10 min. Plasma was stored at -80˚C. miRNA was extracted using the NucleoSpin miRNA plasma kit and cfDNA using the QIAmp circulating nucleic acid kit. miRNA and cfDNA were quantified using the fluorometry-based Qubit microRNA Assay Kit and Qubit dsDNA HS Assay kit, respectively. Individual miRNAs were quantified using the Taqman real-time PCR. Copies of TP53 and PIK3CA were quantified by droplet digital PCR (ddPCR). Levels of miRNA were normalized to specimens of the same tube type stored for 4 h. cfDNA was only investigated in specimens stored for 4 h and 7 days.
Summary of Findings:
Total plasma miRNA levels were unaffected by room temperature storage of blood in Streck, Roche, or Norgen tubes for up to 1 week before centrifugation, but plasma miRNA levels were higher when blood was stored for 24 h in PAXgene tubes than when centrifuged after 4 h. The average level of miRNA was lower when blood was collected in Roche tubes (45 ng/mL) than Streck or Norgen tubes (79 and 86 ng/mL respectively). Average levels of miRNA were highest when blood was collected in PAXgene tubes (130 ng/mL). Levels of the hemolysis marker miR-451 were unaffected by storage of blood for 7 days in PAXgene or Norgen tubes but was significantly increased when blood was stored in Streck tubes for 5 or 7 days (P<0.001, both) or Roche tubes for 7 days (P<0.001). The increased hemolysis in specimens stored in Streck tubes for 6 or 7 days or Roche tubes for 7 days compared to 4 h in the same tubes type was confirmed by spectrophotometer; however, peaks at 414 nm were observed in the specimens stored in Norgen or PAXgene tubes for 4 h, indicating a low level of hemolysis in these specimens, regardless of storage duration. miR-200c levels were unaffected by blood storage duration, regardless of tube type.
cfDNA levels were unaffected by room temperature storage of blood in Streck, Roche, Norgen, or PAXgene tubes for up to 1 week before centrifugation. DNA yields were highest when blood was collected in Norgen tubes (6.9 ng/mL) followed by PAXgene (5.5 ng/mL), Roche (3.9 ng/mL), and Streck (3.6 ng/mL). The DNA peak size was 168-180 bp for specimens collected in PAXgene, Streck, and Roche tubes but 150 bp for those collected in Norgen tubes (P<0.01, versus each of the other tubes). While the number of copies of PIK3CA was comparable between tube types, the counts of TP53 were lower when specimens were collected in Norgen tubes than the other tube types, but significance was not tested.
Biospecimens
Preservative Types
- Streck/BCT
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform DNA Fluorometry DNA Digital PCR Protein Spectrophotometry RNA Real-time qRT-PCR RNA Fluorometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Type of collection container/solution Roche
PAXgene
Streck
Norgen
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
Digital PCR Specific Targeted nucleic acid TP53
PIK3CA
Real-time qRT-PCR Specific Targeted nucleic acid miR 451a
miR-200c
miR-16
miR-376c
Storage Time at room temperature 4 h
24 h
5 days
7 days