Stability of endogenous and added RNA in blood specimens, serum, and plasma.
Author(s): Tsui NB, Ng EK, Lo YM
Publication: Clin Chem, 2002, Vol. 48, Page 1647-53
PubMed ID: 12324479 PubMed Review Paper? No
Suggested by: ISBER
Purpose of Paper
The purpose of this paper was to determine the effects of delayed processing, filtration and freeze-thaw cycling on nucleic acid content of EDTA-plasma and serum.
Conclusion of Paper
Filtration of plasma through a 5 uM filter did not affect glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA levels, but levels declined in plasma with further decreases in filter size. Storage of EDTA-blood or clotted blood at room temperature led to increased GAPDH mRNA levels in unfiltered EDTA-plasma and increased filtered and unfiltered serum beta-globin (b-globin) levels, but did not affect GAPDH mRNA levels in filtered EDTA-plasma or b-globin DNA levels in filtered or unfiltered EDTA-plasma. Storage at 4 degrees C did not affect GAPDH mRNA levels in filtered or unfiltered EDTA-blood or filtered, clotted blood, but GAPDH mRNA and b-globin DNA increased in unfiltered, clotted blood stored at 4 degrees C. Regardless of filter size, there was no effect of 1 freeze-thaw cycle and subsequent storage at room temperature for 1 h on GAPDH mRNA or b-globin DNA levels in serum or plasma. Plasma spiked with RNA showed rapid declines in RNA recovery, and after 15 sec, 99% of the RNA was not amplifiable.
Studies
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Study Purpose
The purpose of this study was to determine the effects of delayed processing, filtration and freeze-thaw cycling on nucleic acid content of EDTA-plasma and serum. 20 EDTA-blood and 20 clotted blood specimens from healthy volunteers were stored at room temperature and 4°C for 0, 6 and 24 h before centrifugation and filtration. Additionally, plasma and serum from 16 healthy patients were divided into thirds for freezing/thawing experiments in which plasma was snap-frozen, but serum was frozen at -20°C. RNA was extracted using RNeasy and DNA was extracted using the QIAamp blood mini-kit. RNA and DNA were quantified by real-time qPCR amplification of GAPDH and β-globin, respectively.
Summary of Findings:
Filtration of plasma through a 5 uM filter did not affect GAPDH mRNA levels, but levels declined in plasma with further decreases in filter size. When EDTA-blood was left at room temperature, GAPDH mRNA levels increased in unfiltered plasma but were not affected when unfiltered plasma was stored at 4 degrees C or when filtered (0.22 uM) plasma was stored at either temperature. B-globin DNA levels in plasma were unaffected by storage of filtered (0.22 uM) or unfiltered EDTA-blood at room temperature or 4 degrees C for up to 24 h. When clotted blood was left at room temperature, GAPDH mRNA levels in serum increased after 6 h but declined slightly (still above baseline) with longer storage. When clotted blood was stored at 4 degrees C, GAPDH levels in serum were higher after 24 h compared to baseline. Filtration (0.22 uM) removed the effects of room temperature and 4 degrees C storage of clotted blood on levels of GAPDH mRNA in serum. Interestingly, serum b-globin DNA levels increased when clotted blood was stored unfiltered at 4 degrees C for 24h or at room temperature, filtered or unfiltered, for 24 or 6 h, respectively. Regardless of filter size, there was no effect of 1 freeze-thaw cycle and subsequent storage at room temperature for 1 h on GAPDH mRNA and b-globin DNA levels in serum or plasma.
Biospecimens
Preservative Types
- None (Fresh)
Diagnoses:
- Normal
Platform:
Analyte Technology Platform RNA Real-time qRT-PCR DNA Real-time qPCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Blood and blood products Plasma
Serum
Storage Freeze/thaw cycling 0 cycles
1 cycle
Storage Post-thaw duration 0 h
1 h
Storage Storage duration 0 h
6 h
24 h
Storage Storage temperature 4 degrees C
Room temperature
Biospecimen Aliquots and Components Filtration None
5 uM
0.45 uM
0.22 uM
Real-time qPCR Specific Targeted nucleic acid b-globin
Real-time qRT-PCR Specific Targeted nucleic acid GAPDH
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
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Study Purpose
The purpose of this study was to determine the effect of room temperature storage on the concentration of exogenous RNA in plasma. 67,605 ng/L of purchased human RNA was added to a single plasma specimen and stored at room temperature for 0, 5, 10 and 15 s before addition of Trizol. RNA was extracted using RNeasy. RNA was quantified by real-time qPCR amplification of GAPDH.
Summary of Findings:
Plasma spiked with RNA showed rapid declines in RNA recovery, and after 15 sec, 99% of the RNA was not amplifiable.
Biospecimens
Preservative Types
- None (Fresh)
Diagnoses:
- Normal
Platform:
Analyte Technology Platform RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration 0 sec
5 sec
10 sec
15 sec