NIH, National Cancer Institute, Division of Cancer Treatment and Diagnosis (DCTD) NIH - National Institutes of Health National Cancer Institute DCTD - Division of Cancer Treatment and Diagnosis

Evaluating transport conditions of conventional, widely used EDTA blood tubes for gene expression analysis in comparison to expensive, specialized PAXgene tubes in preparedness for radiological and nuclear events.

Author(s): Schüle S, Ostheim P, Muhtadi R, Stewart S, Kaletka G, Hermann C, Port M, Abend M

Publication: Int J Radiat Biol, 2023, Vol. , Page 1-9

PubMed ID: 37676284 PubMed Review Paper? No

Purpose of Paper

This paper compared yield, integrity, and expression of radio-sensitive genes in RNA isolated from blood stored for up to 4 h at room temperature; stored for up to 4 h at room temperature followed by storage for 24 h at room temperature, 4°C, or -20°C; and blood that was irradiated before storage.

Conclusion of Paper

Storage of blood at room temperature for ≤ 4 h before transfer to PAXgene tubes or subsequent storage for 24 h at room temperature or 4°C had no effect on RNA yield, RNA integrity numbers (RIN), quantification cycle (Cq) for 18S rRNA, or normalized expression of the investigated radio-sensitive genes (FDXR, DDB2, WNT3, and POU2AF1). However, when blood was stored at room temperature for 4 h and then stored at -20°C for 24 h before being transferred to PAXgene tubes the RIN was lower, 18S rRNA Cq values were higher, and POU2AF1 levels were >2-fold higher  relative to values from blood that was immediately transferred to PAXgene tubes. When blood was only stored at -20°C for 24 h before transfer (no prior room temperature storage) a non-significant reduction in RIN and an increase in 18S rRNA Cq values were observed. RNA yield, RIN, and 18S rRNA values were comparable in RNA extracted from irradiated specimens (stored at 37°C for 8 h, followed by 0 or 4 h at room temperature and then 24 h at room temperature or 4°C) and non-irradiated specimens (stored at 37°C for 8 h). However, normalized expression of FDXR and DDB were much higher (33- to 61-fold and 9- to 17-fold higher, respectively) in irradiated specimens regardless of storage duration and temperature relative to non-irradiated specimens that were immediately transferred to PAXGene tubes, but statistical significance was limited to FDXR expression in irradiated specimens that were stored for 4 h at room temperature followed by 24 h at room temperature. Normalized expression of POU2AF1 and WNT3 were generally lower in irradiated specimens relative to non-irradiated specimens that were immediately transferred to PAXGene tubes, but while the fold changes in POU2AF1 were >2-fold they were not statistically significant and statistically significant changes in WNT3 were mostly < than 2-fold.  

Studies

  1. Study Purpose

    This study compared yield, integrity, and expression of radio-sensitive genes in RNA isolated from blood stored for up to 4 h at room temperature; stored for up to 4 h at room temperature followed by storage for 24 h at room temperature, 4°C, or -20°C; and blood that was irradiated before storage. Blood was collected from 11 healthy volunteers into EDTA tubes.  As a control for all experiments, an aliquot of blood was immediately transferred to PAXgene tubes. To investigate the effect of storage, blood from 3 volunteers was stored for 0, 1, 2, 3, or 4 h at room temperature before transfer to PAXgene tubes. To investigate the effects of storage and shipment, blood from 3 volunteers was stored for 0 or 4 h at room temperature before storage at room temperature, 4°C, or -20°C for 24 h (simulated shipping). To investigate the impact of ionizing radiation and shipment, blood was exposed to X-rays filtered with 3 mm beryllium and 3 mm aluminum to give a mean photon energy of 100 keV (approximately 1.0 gy/h at 13 mA) at 37°C and then stored at 37°C for 8 h followed by 0 or 4 h of storage at room temperature, and then 24 h at room temperature or 4°C. After experimental storage, specimens were transferred to PAXgene tubes and stored at room temperature for 2 h before transfer to -20°C. Before RNA extraction, blood was thawed and cells were pelleted by centrifugation (conditions not specified). RNA was extracted from the pellet using the QIAsymphony PAXgene Blood RNA Kit and a QIAsymphony SP instrument and stored at -20°C. RNA was quantified by spectrophotometer and fragment size was profiled using a 4200 TapeStation instrument. Levels of DXR, DDB2, POU2AF1, and WNT3 were quantified by real-time PCR and normalized to 18S rRNA. Differential expression was defined as a fold change of ≤0.5 or ≥2 and a p-value <0.05.

    Summary of Findings:

    The yield, RIN, and CT values of the 4 RNAs investigated were unaffected by storage of blood at room temperature for ≤ 4 h before being transferred to PAXgene tubes. The RIN was lower, 18S rRNA Cq values higher, and POU2AF1 levels >2-fold higher when blood was stored at room temperature for 4 h and then at -20°C for 24 h before being transferred to PAXgene tubes compared to values from blood that was immediately transferred to PAXgene tubes (P=0.049, P=0.001, and P<0.05 respectively). A non-significant reduction in RIN and an increase in 18S rRNA Cq values was observed when blood was stored at -20°C for 24 h before being transferred to PAXgene tubes.  RNA yield, RIN, and 18S rRNA Cq values were comparable among blood specimens that were immediately transferred and those that were stored for 0 or 4 h at room temperature followed by 24 h at room temperature or 4°C before transfer. Importantly, storage-associated changes in WNT3, DDB2, FDXR, and POU2AF1 were less than 2-fold with the exceptions of WNT3 in blood stored for 24 h at -20°C (not significant) and the aforementioned significant increase in POU2AF1 in blood stored at room temperature for 4 h followed by storage at -20°C for 24 h. RNA yield, RIN, and 18S rRNA values were comparable in RNA extracted from irradiated specimens (stored at 37°C for 8 h, followed by 0 or 4 h at room temperature, and 24 h at room temperature or 4°C) and non-irradiated specimens (stored at 37°C for 8 h). However, normalized expression of FDXR and DDB were much higher (33- to 61-fold and 9- to 17-fold higher, respectively) in irradiated specimens regardless of storage relative to non-irradiated specimens that were immediately transferred to PAXGene tubes, but statistical significance was limited to FDXR in irradiated specimens stored for 4 h at room temperature followed by 24 h at room temperature. Normalized expression of POU2AF1 and WNT3 were generally lower in irradiated specimens relative to non-irradiated specimens that were immediately transferred to PAXgene tubes; but while fold changes in POU2AF1 were >2-fold, they were not statistically significant, and statistically significant changes in WNT3 were mostly < than 2-fold.  

    Biospecimens
    Preservative Types
    • PAXgene
    • Frozen
    Diagnoses:
    • Normal
    Platform:
    AnalyteTechnology Platform
    RNA Spectrophotometry
    RNA Real-time qRT-PCR
    RNA Automated electrophoresis/Bioanalyzer
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Storage Time at room temperature 0 h
    1 h
    2 h
    3 h
    4 h
    Real-time qRT-PCR Specific Targeted nucleic acid WNT3
    DDB2
    FDXR
    POU2AF1
    18S rRNA
    Storage Storage conditions Irradiated
    Not irradiated
    Storage Storage duration 0 h at room temperature followed by 24 h at room temperature
    4 h at room temperature followed by 24 h at room temperature
    0 h at room temperature followed by 24 h at 4°C
    4 h at room temperature followed by 24 h at 4°C
    0 h at room temperature followed by 24 h at -20°C
    4 h at room temperature followed by 24 h at -20°C

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