Quality Control of RNA Extracted from PAXgene® Blood RNA Tubes after Long-Term Cryopreservation.
Author(s): Tang R, Liu J, Wang X, Zhu L, Yin R, Zhu P, Zheng C
Publication: Biopreserv Biobank, 2025, Vol. , Page
PubMed ID: 41161697 PubMed Review Paper? No
Purpose of Paper
This paper compared the yield, purity and integrity of RNA extracted from 217 unmatched PAXgene blood specimens that were stored frozen (-80°C) for 7, 9 or 11 years before extraction. The authors also investigated if storage duration affects the incidence of clot formation using 517 PAXgene blood specimens that were stored for 1 month to 11 years and compared the yield and integrity of RNA extracted from clotted and non-clotted specimens.
Conclusion of Paper
The RNA yield and ratio of absorbance at 260 nm to 230 nm (A260/A230) were comparable among PAXgene blood that was stored frozen (-80°C) for 7, 9 and 11 years. The A260/A280 was greater or equal to the threshold (of 1.8) for all RNA samples but was significantly affected by the duration of PAXgene blood storage. RNA from blood stored for 7 years had a significantly lower median A260/A280 than that from blood stored for 9 years (2.03 versus 2.08, P<0.001) or 11 years (2.03 versus 2.07, P<0.05), and the A260/A280 was higher in RNA from blood stored for 9 years than 11 years (P<0.05). The median RNA integrity number (RIN) value was slightly higher for RNA extracted from blood after 9 years of storage than 11 years (9.0 versus 8.8, P<0.05), but RINs did not differ between PAXgene blood stored 7 versus 9 or 11 years. There was no difference in RIN pass rate (RIN ≥6 ) when PAXgene blood was stored for 7, 9 or 11 years (97%, 98%, and 98%, respectively) or when RIN pass rate was set at a more stringent cut-off of ≥7 (95%, 94%, and 93%, respectively) or ≥8 (87%, 89%, and 86%, respectively). When all 517 specimens were considered, 9% had clots, with a trend of more clots with longer storage (2% for specimens stored 1, 24 or 36 months, 4% for specimens stored 12 months, 12% for specimens stored 72 months, 14% for specimens stored 84 months, 16% for specimens stored 108 months and 9% for specimens stored 132 months). Storage duration was significantly correlated with an incidence of blood clot formation (r=0.795, P<0.05). Median RNA yields were comparable when RNA was extracted from clotted and non-clotted blood (5.15 µg versus 5.29 µg), but the median RIN was lower for clotted blood specimens (6.9 versus 8.6, P<0.001). The authors conclude that PAXgene blood tubes preserve RNA during prolonged frozen storage of blood.
Studies
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Study Purpose
This study compared the yield, purity and integrity of RNA that was extracted from 217 unmatched PAXgene blood specimens stored frozen (-80°C) for 7, 9 or 11 years before extraction. The authors also investigated whether storage duration affects the incidence of clot formation using 517 PAXgene blood specimens stored for 1 month to 11 years and compared the yield and integrity of RNA extracted from clotted and non-clotted specimens. A total of 217 blood specimens were collected from patients with chronic kidney disease into PAXgene Blood RNA tubes and stored at -80°C for 7 years (62 specimens), 9 years (98 specimens) or 11 years (57 specimens). Blood was thawed for 2 h at room temperature with gentle inversion before manual RNA extraction using the PAXgene Blood RNA Kit with an on-column DNAse I treatment step. RNA was quantified using a Qubit Fluorometer and purity assessed by spectrophotometer. RNA integrity was evaluated using a bioanalyzer. To investigate if storage duration affects the incidence of blood clot formation, an additional 300 PAXgene blood specimens stored for 1 month (50 specimens), 12 months (50 specimens), 24 months (50 specimens), 36 months (50 specimens), 72 months (50 specimens), and 84 months (50 additional specimens) were considered in addition to the initial 217 specimens.
Summary of Findings:
The RNA yield and ratio of absorbance at 260 nm to 230 nm (A260/A230) were comparable among PAXgene blood that was stored frozen (-80°C) for 7, 9 and 11 years. The A260/A280 was greater or equal to the threshold (of 1.8) for all RNA samples but was significantly affected by the duration of PAXgene blood storage. RNA from blood stored for 7 years had a significantly lower median A260/A280 than that from blood stored for 9 years (2.03 versus 2.08, P<0.001) or 11 years (2.03 versus 2.07, P<0.05), and the A260/A280 was higher in RNA from blood stored for 9 years than 11 years (P<0.05). The median RNA integrity number (RIN) value was slightly higher for RNA extracted from blood after 9 years of storage than 11 years (9.0 versus 8.8, P<0.05), but RINs did not differ between PAXgene blood stored 7 versus 9 or 11 years. There was no difference in RIN pass rate (RIN ≥6 ) when PAXgene blood was stored for 7, 9 or 11 years (97%, 98%, and 98%, respectively) or when RIN pass rate was set at a more stringent cut-off of ≥7 (95%, 94%, and 93%, respectively) or ≥8 (87%, 89%, and 86%, respectively). When all 517 specimens were considered, 9% had clots, with a trend of more clots with longer storage (2% for specimens stored 1, 24 or 36 months, 4% for specimens stored 12 months, 12% for specimens stored 72 months, 14% for specimens stored 84 months, 16% for specimens stored 108 months and 9% for specimens stored 132 months). Storage duration was significantly correlated with an incidence of blood clot formation (r=0.795, P<0.05). Median RNA yields were comparable when RNA was extracted from clotted and non-clotted blood (5.15 µg versus 5.29 µg), but the median RIN was lower for clotted blood specimens (6.9 versus 8.6, P<0.001). The authors conclude that PAXgene blood tubes preserve RNA during prolonged frozen storage of blood.
Biospecimens
Preservative Types
- PAXgene
Diagnoses:
- Other diagnoses
Platform:
Analyte Technology Platform RNA Spectrophotometry RNA Fluorometry RNA Automated electrophoresis/Bioanalyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration 7 years
9 years
11 years
1 month
12 months
24 months
36 months
72 months
84 months
108 months
132 months
Biospecimen Aliquots and Components Blood and blood products Clotted specimen
Non clotted specimen