Stabilization of cellular RNA in blood during storage at room temperature: a comparison of cell-free RNA BCT(®) with K3EDTA tubes.
Author(s): Das K, Norton SE, Alt JR, Krzyzanowski GD, Williams TL, Fernando MR
Publication: Mol Diagn Ther, 2014, Vol. 18, Page 647-53
PubMed ID: 25178583 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to determine the effects of blood collection tube type and delayed processing on neutrophil mRNA yields and purity, and levels of proto-oncogene c-Fos (c-Fos), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and tumor protein p53 (p53).
Conclusion of Paper
Storing blood for 3 days at room temperature in Streck blood collection tubes (BCT) or EDTA tubes did not significantly affect neutrophil mRNA yields and purity, or c-Fos mRNA levels. Although GAPDH and p53 mRNA levels were comparable in immediately processed specimens collected in EDTA and BCT tubes, c-Fos mRNA levels were lower when the blood was collected in BCT tubes rather than EDTA tubes. Storage of blood at room temperature for up to 3 days in BCT tubes did not affect mRNA levels of GAPDH, p53 or c-Fos; but significant decreases in p53 and increases in GAPDH mRNA occurred when blood was stored in EDTA tubes for 1 day.
Studies
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Study Purpose
The purpose of this study was to determine the effects of blood collection tube type and delayed processing on neutrophil mRNA yields and purity, and levels of c-Fos, GAPDH, and p53. Blood from 6 healthy donors was collected into 4 EDTA and 4 BCT tubes. After the relevant room temperature storage, specimens were centrifuged, red blood cells were lysed, and neutrophils were isolated using CD15 MicroBeads. Neutrophils were counted and then stored at -80˚C until RNA extraction using the RNeasy FFPE kit. mRNA levels were quantified by real-time PCR and normalized to neutrophil counts.
Summary of Findings:
Storing blood for 3 days at room temperature in Streck blood collection tubes (BCT) or EDTA tubes did not significantly affect neutrophil mRNA yields or purity (OD 160/280 and OD 260/230 ratios). Although neutrophil GAPDH and p53 mRNA levels were comparable in immediately processed specimens collected in EDTA and BCT tubes, c-Fos mRNA levels were lower when blood was collected in BCT tubes rather than EDTA tubes. Storage of blood at room temperature for up to 3 days in BCT tubes did not affect mRNA levels of GAPDH, p53 or c-Fos. When blood was stored at room temperature in EDTA tubes for 1 day rather than processed immediately, neutrophil copies of GAPDH mRNA increased 5.8-fold (p=0.01) and neutrophil copies of p53 mRNA decreased 2.4-fold (p=0.005). A similar 3.2-fold decrease in neutrophil p53 mRNA levels was observed when blood was stored at room temperature in EDTA tubes for 2 days rather than processed immediately (p=0.04), but storage of blood in EDTA tubes at room temperature for 3 days did not significantly affect GAPDH or p53 levels. Neutrophil c-Fos mRNA levels were not affected by room temperature storage of blood.
Biospecimens
Preservative Types
- Frozen
- Streck/BCT
Diagnoses:
- Normal
Platform:
Analyte Technology Platform RNA Spectrophotometry RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Type of collection container/solution BCT tube
EDTA tube
Biospecimen Preservation Type of fixation/preservation Blood collection tube additive
None (fresh)
Storage Time at room temperature 0 days
1 day
2 days
3 days
Real-time qRT-PCR Specific Targeted nucleic acid c-Fos
p53
GAPDH