Collection and storage of human blood cells for mRNA expression profiling: a 15-month stability study.
Author(s): Marteau JB, Mohr S, Pfister M, Visvikis-Siest S
Publication: Clin Chem, 2005, Vol. 51, Page 1250-2
PubMed ID: 15976103 PubMed Review Paper? No
Purpose of Paper
Conclusion of Paper
Studies
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Study Purpose
The purpose of this study was to determine the effects of anticoagulant type, lysis solution, and storage of lysates at -70 degrees C on the yield and integrity of RNA from PBMC. RNA was extracted from the cell lysates using the MagNa Pure LC RNA isolation kit and eluted at 70 degrees C.
Summary of Findings:
The type of anticoagulant used had no effects on A260/A280 ratios, but yields were higher from blood anticoagulated with EDTA rather than heparin as determined by spectrophotometer (23.9 versus 19.1 ng/uL p<0.0001) and real-time RT-PCR amplification of Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (38 versus 40.4 cycles, p<0.0001). Generally, the yields were highest when the PBMC were lysed using the InstaPure solution rather than when PBMC were lysed using solutions from the other kits, but this was not the case in heparinated PBMC when yields were assessed by amplification of adrenomedullin (ADM) or interleukin-8 (IL-8). Storing cell lysates at -70 degrees C for up to 15 months prior to extraction had no effects on the yield or integrity of RNA.
Biospecimens
Preservative Types
- None (Fresh)
- Frozen
Diagnoses:
- Not specified
Platform:
Analyte Technology Platform RNA Spectrophotometry RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Analyte Extraction and Purification Cell/tissue permeabilization RNA InstaPure
MagNa Pure lysis/binding buffer
QIAamp RTL buffer
StrataPrep RNA lysis buffer
Storage Storage temperature 0 months
3 months
5 months
10 months
15 months
Biospecimen Acquisition Anticoagulant EDTA
Heparin
Real-time qRT-PCR Specific Targeted nucleic acid GAPDH
ADM
IL-8
Biospecimen Preservation Type of fixation/preservation Frozen
None (fresh)