Stability of Endogenous and Added RNA in Blood Specimens, Serum, and Plasma

TLDR

Circulating RNA in plasma and serum is an emerging field for noninvasive molecular diagnosis. The study aimed to assess the stability of endogenous and added RNA in blood specimens and the impact of preanalytical variables. Researchers collected blood from 65 healthy volunteers and examined the effects of processing delays, freezing/thawing, and added RNA lability on RNA concentrations in EDTA blood and clotted blood. Plasma RNA remained stable in uncentrifuged EDTA blood stored at 4 °C for up to 24 h, whereas serum RNA increased over 24 h in uncentrifuged clotted blood; added RNA was largely degraded within 15 s, and a single freeze/thaw cycle had no significant effect on RNA levels, indicating that plasma RNA is stable under these conditions and that serum RNA requires prompt processing within 6 h.

Abstract

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.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.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).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.