RNA quality and gene expression analysis of ovarian tumor tissue undergoing repeated thaw-freezing.
Author(s): Jochumsen KM, Tan Q, Dahlgaard J, Kruse TA, Mogensen O
Publication: Exp Mol Pathol, 2007, Vol. 82, Page 95-102
PubMed ID: 16842777 PubMed Review Paper? No
Purpose of Paper
The paper investigated the effects of repeated freeze-thaw events on RNA quality and quantity and gene expression profiles of ovarian tumor tissues.
Conclusion of Paper
Repeated freeze-thaw cycles had no effect on RNA quantity or RNA quality as determined by RNA Integrity Number (RIN) and degradation factor. In addition, microarray analysis revealed no significant differences due to freeze-thawing in gene expression profiles of the specimens tested. The percentage of specimens that had an acceptable amount of RNA (≥ 90 µg) that was of high quality (RIN ≥ 7.5) did differ between specimens stored at -80ºC for 1 year (88%) and those stored for 8 to 13 years (50%), although the authors note differences in warm ischemia and processing regimes may be confounding variables.
Studies
-
Study Purpose
The study investigated the effects of repeated freeze-thaw events on RNA quality and quantity and gene expression profiles using eight ovarian tumor specimens collected from 1992 to 1997 (“old”) and eight tumor specimens collected from 2004–2005 (“new”). All specimens were stored at -80°C. Each OCT-embedded specimen was placed on a -20°C marble board for 30 sec to 2 min while the it was divided in three equal aliquots during three separate events, from which RNA was extracted and purified. The remaining portion of the OCT-embedded specimen was re-frozen to −80°C between events, which occurred on different days. RNA was stored at -80°C after extraction, measured spectrophotometrically for yield, and by bioanalyzer to determine RNA Integrity Number (RIN) and degradation factor. Four specimens from the “old” specimen set with high or acceptable RNA quality in all three aliquots were analyzed by microarray for gene expression profiles.
Summary of Findings:
Three of the eight “old” tumor specimens had no detectable RNA for any of the three aliquots and one specimen had measurable amounts of RNA (≥ 90 µg) but with low RIN (0 – 3.0) and high degradation factor (> 50) for all three aliquots. The remaining four “old” tumor specimens had acceptable amounts of RNA in all aliquots but high quality RNA in three specimens (RIN = 8.2 – 9.5) and low quality for one specimen (RIN = 4.9 – 8.3). Seven of the “new” tumor specimens had acceptable amounts of RNA and were of high quality as determined by high RIN (≥ 7.5) and low degradation factor (< 50). One “new” tumor contained small amounts of RNA in the second and third day aliquots which the authors attribute to the fact that the specimen was composed of coagulated blood rather than tumor tissue. Decreased RNA yields observed in “old” specimens were attributed to warm ischemia or improper handling before or during freezing. Hierarchical cluster analysis of the gene expression profiles revealed no significant effects due to repeated freeze-thaw cycles. Further, analysis using interclass correlation coefficient (ICC) reveals high correlation between the three aliquots for each specimen (ICC = 0.986) but a significantly lower correlation between the four specimens (ICC = 0.832), indicating that the variability observed between patients was greater than that observed between aliquots subjected to different numbers of freeze-thaw events.
Biospecimens
Preservative Types
- Frozen
- OCT
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform RNA Automated electrophoresis/Bioanalyzer RNA Spectrophotometry RNA DNA microarray Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration 1 y
8-13 y
Storage Freeze/thaw cycling 1 cycle
2 cycles
3 cycles