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Study for evaluating the effect of cold ischemic time and RNA stabilization method on RNA integrity and gene expression measurements

ABSTRACT: Time series of eleven breast cancer samples subjected to different cold ischemic stress of up to 3 hr post tumor excision. A different 2x2 factorial within this study evaluated the effect of stabilization method (RNAlater vs snap freezing) and stablization delay (0 and 40 min) at room temperature. Tissue samples were collected at surgery, cut into 1-2 mm pieces and divided into 8 portions. Portions were put in RNAlater or fresh frozen at baseline, 20, 40, 60, 120, and 180 minutes thereafter, or snap frozen in dry ice in a pre-chilled sample vial at baseline and 40 minutes thereafter.

ORGANISM(S): Homo sapiens

SUBMITTER: Hatzis C

PROVIDER: S-ECPF-GEOD-25011 | biostudies-other | 2011 Dec

REPOSITORIES: biostudies-other

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Project description:BackgroundReliable stability and yield of RNA from breast cancer tissues are important for biobanking, clinical trials, and diagnostic testing.MethodsAliquots of fresh primary tumor tissue from 17 surgically resected invasive breast cancers were placed into RNAlater at room temperature after tumor removal (baseline) and up to 3 hours thereafter or were snap frozen at baseline and 40 minutes thereafter. Samples were stored at -80°C until gene expression profiling with Affymetrix Human Gene U133A microarrays. We evaluated the effects of cold ischemic time (the time from tumor specimen removal to sample preservation) and sample preservation method on RNA yield, Bioanalyzer-based RNA integrity number, microarray-based 3'/5' expression ratios for assessing transcript integrity, and microarray-based measurement of single-gene and multigene expression signatures. The statistical significance of the effects was assessed using linear mixed effects regression models. All statistical tests were two-sided.ResultsSample preservation in RNAlater statistically significantly improved RNA integrity compared with snap freezing as assessed by the RNA integrity number, which increased from 7.31 to 8.13 units (difference = 0.82 units, 95% confidence interval [CI] = 0.53 to 1.11 units, P < .001), and RNA yield, which increased threefold from 8.9 to 28.6 μg (difference = 19.7 μg, 95% CI = 14.1 to 25.3 μg, P < .001). Prolonged cold ischemic delay at room temperature before sample stabilization decreased the RNA integrity number by 0.12 units/h (95% CI = 0.02 to 0.23 units/h) compared with a projected average RNA integrity number of 7.39 if no delays were present (P = .008) and decreased the RNA yield by 1.5 μg/h (95% CI = 0 to 4 μg/h) from a baseline mean RNA yield of 33.5 μg if no delays were present (P = .019). Prolonged cold ischemia statistically significantly increased the 3'/5' ratio of control gene transcripts, particularly of STAT1 (P < .001). Snap freezing statistically significantly increased the 3'/5' ratio of three control transcripts (ACTB, GAPDH, and 18S rRNA). Expression levels of single genes and multigene signatures for breast cancer were largely unaffected by sample preservation method or cold ischemia.ConclusionsSample preservation in RNAlater improves RNA yield and quality, whereas cold ischemia increases RNA fragmentation as measured by the 3'/5' expression ratio of control genes. However, expression levels of single genes and multigene signatures that are of diagnostic relevance in breast cancer were mostly unaffected by sample preservation method or prolonged cold ischemic duration.

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Study for evaluating the effect of cold ischemic time and RNA stabilization method on RNA integrity and gene expression measurements

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