Effects of Transport and Storage Conditions on Gene Expression in Blood Samples.
Author(s): Malentacchi F, Pizzamiglio S, Wyrich R, Verderio P, Ciniselli C, Pazzagli M, Gelmini S
Publication: Biopreserv Biobank, 2016, Vol. 14, Page 122-8
PubMed ID: 26886447 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of shipping blood on gel packs or at ambient temperature and of storing blood at room temperature (RT) and 4°C on RNA yield and quality and gene expression profiles of blood in PAXgene and EDTA tubes.
Conclusion of Paper
RNA yields varied across all storage durations at both temperatures whether shipped or maintained at the collection laboratory but no significant trend was observed. All specimens had RNA integrity numbers (RIN) values >5 except the PAXgene specimens not shipped and stored at RT for 4 days. Gene expression profiles were not affected by shipment on gel packs or at ambient temperature or storage for up to four days at either temperature in PAXgene tubes. In contrast, gene expression levels were significantly affected in EDTA specimens by shipping and all storage conditions.
Studies
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Study Purpose
This study investigated the effects of shipping and storage duration at RT and 4°C on RNA yield and quality and gene expression profiles of blood in PAXgene and EDTA tubes. Whole blood was collected from a single donor into a blood collection bag containing K3EDTA, transferred to a sterilized flask, mixed while being cooled on wet ice, and then immediately aliquoted into EDTA (n=34) and PAXgene Blood RNA (n=34) tubes. RNA was extracted using the PAXgene Blood RNA kit from one tube of each type and then half of the remaining specimens were stored at RT or 4°C at the collection laboratory and the other half were shipped via an international courier to a second laboratory either at ambient temperature or with gel packs specific for preservation at 2–8°C for 48 h. Shipped specimens were stored at RT or in the shipping box with gel packs (uncontrolled temperature). RNA was extracted after 1, 2, 3, or 4 days of storage at each site using the PAXgene Blood RNA kit. RNA yield was determined by Nanodrop and RNA integrity numbers (RINs) were assigned using a bioanalyzer. Gene expression levels of c-FOS, IL-1b, IL-8, TNF, LMNA, FOSB, TNRFSF10c, and GAPDH were determined by real-time qRT-PCR.
Summary of Findings:
RNA yields varied across all storage durations at both temperatures whether shipped or maintained at the collection laboratory but no significant trend was observed. All specimens had RIN values >5 except the PAXgene specimens stored at RT for 4 days (not shipped). Gene expression profiles were not affected by shipment (at ambient temperature or with gel packs at 2-8°C) or storage for up to four days at RT or with gel packs in PAXgene tubes compared to specimens processed immediately after collection. For EDTA tubes, only TNF levels were affected by shipment at 4°C (P=0.0006) but all genes except GAPDH and TNFRSF10c were affected by shipment at ambient temperature (P<0.01 for c-FOS, IL-1β, IL-8, TNF, LMNA, and FOSB). As expected, GAPDH gene expression was not statistically affected by storage at either RT or 4°C in EDTA tubes. c-FOS gene expression did not change over time when stored at 4°C for up to four days in non-shipped EDTA specimens but decreased slightly when stored at ambient (shipped) or RT non-shipped specimens (P≤0.006 all). IL1β expression was statistically different in EDTA blood stored at RT (P=0.0003) or 4°C (P<0.0001) and in specimens shipped at ambient temperature and then stored at RT or with gel packs at an uncontrolled temperature (P<0.0001 both). IL8 transcript levels were not statistically different over time in EDTA specimens stored at 4°C but increased significantly when stored at RT (P≤0.0001) and increased in the shipped specimens stored either at ambient or uncontrolled temperature on gel packs (P≤0.0001 both). LMNA, TNF, and FOSB levels significantly increased over time in EDTA blood stored at RT or shipped and stored both at RT and uncontrolled temperature (P<0.001) but no significant change was observed in specimens stored at 4°C. TNFRSF10c levels were not statistically different in EDTA blood stored at 4°C without shipping but significantly decreased when stored at RT or when shipped (both at ambient or with gel packs) and then stored at room temperature (P<0.0001 for all).
Biospecimens
Preservative Types
- PAXgene
- None (Fresh)
Diagnoses:
- Normal
Platform:
Analyte Technology Platform RNA Automated electrophoresis/Bioanalyzer RNA Spectrophotometry RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Type of collection container/solution PAXgene
EDTA
Biospecimen Preservation Type of fixation/preservation PAXgene
None (fresh)
Storage Storage duration 1 day
2 days
3 days
4 days
Real-time qRT-PCR Specific Targeted nucleic acid c-FOS
IL-1β
IL-8
TNF
LMNA
FOSB
TNRFSF10c
GAPDH
Storage Between site transportation method Not transported
Air courier
Storage Specimen transport duration/condition Ambient Temperature
With gel packs specific for preservation at 2–8°C for 48 h
Storage Storage temperature Room temperature
4°C
With gel packs specific for preservation at 2–8°C for 48 h