Your Blood is Out for Delivery: Considerations of Shipping Time and Temperature on Degradation of RNA from Stabilized Whole Blood.
Author(s): Stefanovic F, Brown LG, MacDonald J, Bammler T, Rinchai D, Nguyen S, Zeng Y, Shinkawa V, Adams K, Chaussabel D, Berthier E, Haack AJ, Theberge AB
Publication: Anal Chem, 2025, Vol. 97, Page 1635-1644
PubMed ID: 39818791 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of storing aliquots of RNAlater-stabilized blood at temperatures that ranged between 25 and 50℃ for up to 8 days on RNA integrity. The effect of an additional overnight storage event either before (4℃ or 25℃ ) or after (25℃) the main storage event, and a single freeze-thaw cycle that included 24 h at room temperature were also investigated. Effects of transport time and temperature on RINs and next-generation sequencing results were investigated by shipping aliquots of RNAlater-stabilized blood round-trip to 14 different states.
Conclusion of Paper
RNA integrity numbers (RIN) declined at higher storage temperatures and longer durations of storage in RNAlater-stabilized blood, but the average RIN remained >7 when RNAlater-stabilized blood was stored at 25℃ for up to 8 days, and RIN remained >3 (author’s threshold for transcriptomic analysis) when specimens were stored at ≤50℃ for up to 2 days. There was no clear effect on RIN of initial overnight storage (16 h) at 4℃ or 25℃ before additional storage, an initial storage duration of 6 h, or a single freeze-thaw cycle that included 24 h at room temperature. During specimen shipment to 14 different states (shipment duration ranged between 3-8 days, with 10 states taking 3 days), temperature probes included within the packages recorded temperatures as high as 45.1℃, with many recording temperatures above 30℃ for as long as 55 h (shipped to Nebraska). The lowest RINs were observed in RNA from RNAlater-stabilized blood aliquots that were shipped to NE (the highest recorded temp and a transport duration of 7 days), Arizona (38.3 h above 30℃, 7 days of transport) and Hawaii (22.3 h above 30℃ and 8 days of transport), but RINs remained above the 3.0 threshold for sequencing. RNAlater-stabilized blood aliquots shipped to California reached a higher temperature than those shipped to Hawaii or Arizona (44.4℃ versus 40℃ and 38.5℃, respectively), but had a shorter transit time (3 days versus 8 days and 7 days, respectively) and a shorter duration above 30℃ (11.1 h versus 22.3 h and 38.3 h, respectively) that resulted in a higher mean RIN (7.0 versus 6.1 and 6.2, respectively); the authors concluded that transport time rather than temperature only is important to RNA integrity. Further indicating the importance of storage duration, the RIN of RNA from RNAlater-stabilized blood aliquots stored in the laboratory for 8 days at 25°C was lower than in the matched aliquots shipped to any of the 11 states that returned within 4 days of transport. Interestingly, Principal component analysis (PCA) of sequencing data clustered the specimens stored in the laboratory separately from shipped (all locations) and control (immediately frozen) specimens, but the variability captured by the first two components was low (16.25% and 8.97%, respectively). Heatmap analysis showed only small differences in enrichment across the modules investigated (interferon, inflammation and circulating erythroid cells signatures), including declines in two transcripts by 34% for every 10 h the RNAlater-stabilized blood spent above 30℃.
Studies
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Study Purpose
This study investigated the potential effects of storing aliquots of RNAlater-stabilized blood at ≤50℃ for up to 8 days on RNA integrity. The effect of an additional overnight storage period either before (4℃ or 25℃ ) or after (25℃) the experimental storage, and a single freeze-thaw cycle that included 24 h at room temperature were also investigated. Effects of transport time and temperature on RIN and next-generation sequencing (NGS) results were investigated by shipping aliquots of RNAlater-stabilized blood round-trip to 14 different states and comparing results to specimens stored in the laboratory for 8 days or frozen immediately. Blood was collected into EDTA Vacutainer tubes from an unspecified number of healthy volunteers and stabilized with RNAlater at the recommended ratio. RNAlater-stabilized blood was aliquoted into 1.8 mL tubes. Aliquots of a single RNAlater-stabilized blood specimen were stored at 25°C, 37°C, 40°C, 45°C, and 50°C for 0, 2, 4 and 8 days either immediately or after overnight storage (16 h) at 4°C or 25°C. To investigate freeze-thaw cycling, matched aliquots of a single RNAlater-stabilized blood specimen were immediately frozen (control), or stored at room temperature for 24 h before freezing or frozen, and then stored at room temperature for 24 h before refreezing (freeze-thaw). To investigate potential effects associated with short-term storage, aliquots of a single RNAlater-stabilized blood specimen were stored overnight (16 h) at 25°C and then either frozen or stored at 25°C, 37°C, 40°C, 45°C, and 50°C for 6 h, after which they were frozen immediately or after an additional 16 h at 25°C. To investigate effects of real-word shipping conditions, aliquots of a single RNAlater-stabilized blood specimen were placed in 50 mL tubes, taped together in triplicate, and placed in a homeRNA box (a self-sampling device) with a continuous temperature monitor and shipped roundtrip to 14 different states (held inside overnight before return shipment) or stored in a 25°incubator for 8 days before freezing. After storage/shipment, all specimens were frozen at -20°C and then moved to -80°C for storage until analysis. RNA was extracted using the Ribopure Blood RNA Isolation Kit and stored at -80°C. RNA integrity was assessed by Bioanalyzer. Sequencing libraries were prepared using the QuantSeq 3’ mRNA-seq V2 Library Prep Kit FWD with UDI and sequenced on an Illumina NextSeq 2000. Sequencing data was analyzed using the limma-voom pipeline in R.
Summary of Findings:
RINs were comparable in RNAlater-stabilized blood specimens that were immediately frozen (control), and those that were stored at room temperature for 24 h before freezing, or frozen and then stored at room temperature for 24 h before refreezing (a freeze-thaw cycle). There was no effect of storing RNAlater-stabilized blood overnight at room temperature before freezing. RIN did decline at higher temperatures and with longer storage duration of RNAlater-stabilized blood, but RINs did not differ in RNAlater-stabilized blood that was stored overnight storage (16 h) at 4℃ versus 25℃ before storage. Further, despite a small decline, the average RIN remained >7 when the RNAlater-stabilized blood was stored at 25℃ for up to 8 days. Using a threshold RIN ≥3, which the authors state is suitable for transcriptomic analysis, all specimens stored for 2 days had suitable RINs regardless of the temperature of storage, but the RIN was higher when blood was stored at 25℃ compared to those stored at 50℃ (RIN of 7.9 versus 5.5). However, when RNAlater-stabilized blood was stored for 4 days at 50℃, some stored specimens had a RIN <3. When specimens were stored overnight at 25℃, followed by 6 h, the mean RIN ranged from 6.4-7.2 across the storage temperatures evaluated (25-50℃), with no clear effect of storage temperature; but RINs as low as 4.9 (observed in a specimen stored at 25℃) were observed. There was no effect of storing the blood specimen at 25℃ overnight after the 6 h storage. During shipment to 14 different states (transport time ranged between 3-8 days, with 10 states taking 3 days), temperature probes in the packages recorded temperatures as high as 45.1℃, with many recording temperatures above 30℃. The lowest RINs (mean 5.5, 6.1 and 6.2, respectively)f rom RNAlater-stabilized blood aliquots were observed in specimens that were shipped to NE (highest recorded temp, 7 days of transport and 55 h above 30℃ ), Arizona (38.3 h above 30℃, 7 days of transport) and Hawaii (22.3 h above 30℃ and 8 days of transport); but RINs remained above the 3.0 threshold for sequencing in all specimens. RNAlater-stabilized blood aliquots shipped to California reached a higher temperature than those shipped to Hawaii or Arizona (44.4℃ versus 40℃ and 38.5℃, respectively), but had a shorter transit time (3 days versus 8 days and 7 days, respectively) and shorter duration above 30℃ (11.1 h versus 22.3 h and 38.3 h, respectively), resulting in a higher mean RIN (7.0 versus 6.1 and 6.2, respectively); the authors state that this indicates that the transport time rather than temperature only is an important consideration. Further indicating the importance of storage duration, the RIN of RNAlater-stabilized blood aliquots stored in the laboratory for 8 days at 25°C was lower than the matched aliquots shipped to any of the 11 states that were returned within 4 days. Interestingly, PCA of sequencing data clustered specimens stored in the laboratory separately from shipped (all locations) and control (immediately frozen) specimens;,but, the variability captured by the first two components was low (16.25% and 8.97%, respectively). Heatmap analysis showed only small differences in enrichment across the modules investigated (interferon, inflammation and circulating erythroid cells signatures). In contrast, the Interferon Modules Enrichment Scores showed clear enrichment following vaccination, which the authors state indicates that the small changes observed are negligible compared to those that occur as a result of biological signaling. However, levels of two transcripts (MMP9 and ADGRG3) were found to decrease by 34% for every 10 h that the RNAlater-stabilized blood spent above 30℃.
Biospecimens
Preservative Types
- RNAlater
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform RNA Next generation sequencing RNA Automated electrophoresis/Bioanalyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage temperature 25°C
37°C
40°C
45°C
50°C
Storage Storage duration 0 h
6 h
0 days
2 days
4 days
8 days
With/Without additional overnight storage (16 h)
Storage Specimen transport duration/condition ≤3 days
4 days
7 days
8 days
Max temp 25.7-44.4°C
0-55 h above 30°C
Storage Freeze/thaw cycling 1 cycle
2 cycles
Storage Between site transportation method Mailed
Not transported