Urine exosome mRNA-based test for monitoring kidney allograft rejection: Effects of sample transportation and storage, and interference substances.
Author(s): McFaul M, Ventura C, Evans S, Dundar H, Rumpler MJ, McCloskey C, Lowe D, Vlassov AV
Publication: World J Methodol, 2023, Vol. 13, Page 492-501
PubMed ID: 38229935 PubMed Review Paper? No
Purpose of Paper
This paper compared levels of nine mRNAs in urine that was stored for 2 days at 20°C or 40°C rather than 4°C, in urine that was freeze-thaw cycled once or twice versus stored at 4°C, in urine stored at 4°C for 7 or 14 days versus 2 days, in urine stored at -80°C for 4 or 30 days rather than fresh urine, and in urine spiked with commonly prescribed medication, blood, or serum.
Conclusion of Paper
Cycle threshold (CT) values were higher when urine was stored for 2 days at 20°C or 40°C or freeze-thaw cycled than when specimens were stored at 4°C, but the magnitude of the difference was dependent on the mRNA target and patient source. The authors noted highly variable CT values when urine was freeze-thaw cycled without prior centrifugation, which they state confirms the necessity of centrifugation to remove contaminating cellular RNA. CT values were comparable in specimens stored at 4°C for 2 days or 7 days, but CT values were higher when urine was stored at 4°C for 14 days. While CT values were higher for frozen compared to fresh urine, no further increase in CT values was observed when urine was stored at -80°C for 30 days versus 4 days. None of the spiked-in drugs affected the CT values for the tested mRNA in four urine specimens. As expected, urine spiked with blood had lower CT values for the mRNAs evaluated than unspiked urine, but when the blood cells were removed by centrifugation before exosome isolation, CT values were comparable to those in unspiked urine. Similarly, urine spiked with serum had comparable CT values to unspiked urine.
Studies
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Study Purpose
This study compared levels of nine mRNAs in urine that was stored for 2 days at 20°C or 40°C rather than 4°C, in urine that was freeze-thaw cycled once or twice versus stored at 4°C, in urine stored at 4°C for 7 or 14 days versus 2 days, in urine stored at -80°C for 4 or 30 days rather than fresh urine, and in urine spiked with commonly prescribed medication, blood, or serum. Urine was collected from four volunteers (details not provided). Exosomes and then RNA were isolated from urine using the ExoLution protocol and silica spin columns. RNA was immediately reverse transcribed using the SuperScript VILO cDNA Synthesis Kit and preamplified using the TaqMan PreAmp Master mix. Nine mRNAs were quantified by real-time PCR. To investigate the effects of storage temperature, urine was stored for 2 days at 4°C, 20°C, or 40°C and freeze-thaw cycled as whole urine or cell-free urine (obtained by centrifugation at 2000 g for 20 min). To investigate the effects of prolonged storage, urine was stored for 2, 7, and 14 days at 4°C or for 4 and 30 days at -80°C. To test if medications commonly prescribed to transplant patients would interfere with the detection of exosomal mRNA in urine, urine was spiked with one of the following medications: 0.53 μg/mL Tacrolimus, 10.5 μg/mL Cyclosporin A, 16.67 μg/mL Mycophenolic acid, 0.03 μg/mL Everolimus, 0.10 μg/mL Sirolimus, 0.47 μg/mL Ascomycin, and 18.33 μg/mL Teriflunomide. To test if blood or serum would interfere with the detection of exosomal mRNA in urine, matched aliquots of urine from four volunteers were left untreated, spiked with 1% blood, spiked with 1% blood and centrifuged at 2000 g for 20 min before processing, or spiked with serum.
Summary of Findings:
CT values were higher when urine was stored for 2 days at 20°C or 40°C or freeze-thaw cycled compared to levels in specimens stored at 4°C, but the magnitude of the difference was dependent on the mRNA target and volunteer source. The authors noted that CT values were highly variable when urine was freeze-thaw cycled without prior centrifugation, which they state confirms the necessity of centrifugation to remove contaminating cellular RNA. CT values were comparable in specimens stored at 4°C for 2 days or 7days, but CT values were higher when urine was stored at 4°C for 14 days. While CT values were higher for frozen than fresh urine specimens, no further increase in CT values was observed when urine was stored at 80°C for 30 days versus 4 days. CT values of the targeted miRNAs were not altered by the presence of any of the medications that were spiked into urine specimens. As expected, urine spiked with blood had lower CT values for the mRNAs evaluated than unspiked urine, but when the blood cells were removed by centrifugation before exosome isolation, CT values were comparable to those in unspiked urine. Similarly, urine spiked with serum had comparable CT values to unspiked urine.
Biospecimens
Preservative Types
- None (Fresh)
- Frozen
Diagnoses:
- Not specified
Platform:
Analyte Technology Platform RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage temperature 4°C
20°C
40°C
-80°C
Storage Storage duration 2 days
7 days
14 days
4 days
30 days
Storage Freeze/thaw cycling 0 cycles
1 cycle
2 cycles
Storage Storage conditions Centrifuged before freezing
Not centrifuged before freezing
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
Centrifuged
Not centrifuged
Biospecimen Aliquots and Components Biospecimen components .53 μg/mL Tacrolimus
10.5 μg/mL Cyclosporin A
16.67 μg/mL Mycophenolic acid
0.03 μg/mL Everolimus
0.10 μg/mL Sirolimus
0.47 μg/mL Ascomycin
18.33 μg/mL Teriflunomide
1% blood
1% serum