Stability of endogenous and added RNA in blood specimens, serum, and plasma.
BACKGROUND
Circulating RNA in plasma/serum is an emerging field for noninvasive molecular diagnosis. Because RNA is widely thought to be labile in the circulation, we investigated the stability and various preanalytical factors that may affect RNA concentrations in blood specimens.


METHODS
Blood samples were collected from 65 healthy volunteers. The effects of two preanalytical variables were studied: (a) time delay in processing of EDTA blood and clotted blood after venesection, and (b) freezing and thawing of plasma and serum. The lability of free added RNA in plasma was also investigated. Plasma/serum RNA was measured by a real-time quantitative reverse transcription-PCR assay for glyceraldehyde 3-phosphate dehydrogenase mRNA, whereas DNA was measured by a real-time quantitative PCR assay for the beta-globin gene.


RESULTS
No significant difference was found for plasma RNA concentrations obtained from uncentrifuged EDTA blood that had been left at 4 degrees C for 0, 6, and 24 h (P =0.182). On the other hand, the serum RNA concentrations increased significantly over 24 h when uncentrifuged clotted blood was stored at 4 degrees C (P <0.05). In comparison, >99% of the free added RNA could no longer be amplified after incubation in plasma for 15 s. Never-frozen plasma, freeze-thawed plasma, and thawed plasma left at room temperature for 1 h showed no significant differences in RNA concentration (P =0.465). No significant difference was observed for freeze-thawed serum (P = 0.430).


CONCLUSIONS
Plasma RNA is stable in uncentrifuged EDTA blood stored at 4 degrees C, but to obtain a stable serum RNA concentration, uncentrifuged clotted blood should be stored at 4 degrees C and processed within 6 h. A single freeze/thaw cycle produces no significant effect on the RNA concentration of plasma or serum.
D001800:Blood Specimen Collection; D004247:DNA / Q000097:blood; D004355:Drug Stability; D005914:Globins / Q000235:genetics; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006801:Humans; D010949:Plasma; D016133:Polymerase Chain Reaction; D012313:RNA / Q000097:blood*; D013997:Time Factors
D012313:RNA; D005914:Globins; D004247:DNA; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases