Effects of Tissue Handling on RNA Integrity and Microarray Measurements From Resected Breast Cancers

• Published in: JNCI : Journal of the National Cancer Institute 2011-12, Vol.103 (24), p.1871-1883
• Main Authors: Hatzis, Christos, Sun, Hongxia, Yao, Hui, Hubbard, Rebekah E., Meric-Bernstam, Funda, Babiera, Gildy V., Wu, Yun, Pusztai, Lajos, Symmans, W. Fraser
• Format: Article
• Language: English
• Subjects: Adult; Aged; Aged, 80 and over; Biological and medical sciences; Biopsy; Breast cancer; Breast Neoplasms - genetics; Breast Neoplasms - pathology; Breast Neoplasms - surgery; Carcinoma, Ductal, Breast - genetics; Carcinoma, Ductal, Breast - pathology; Carcinoma, Ductal, Breast - surgery; Carcinoma, Lobular - genetics; Carcinoma, Squamous Cell - genetics; Cryopreservation - methods; Female; Freeze Drying; Freezing; Gene expression; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gynecology. Andrology. Obstetrics; Humans; Ischemia; Mammary gland diseases; Mastectomy - methods; Medical sciences; Microarray Analysis - methods; Middle Aged; Ribonucleic acid; RNA; RNA, Neoplasm - metabolism; Specimen Handling - adverse effects; Specimen Handling - methods; Time Factors; Tumors
• ISSN: 0027-8874; 1460-2105
• DOI: 10.1093/jnci/djr438

Background Reliable stability and yield of RNA from breast cancer tissues are important for biobanking, clinical trials, and diagnostic testing. Methods Aliquots 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. Results Sample 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. Conclusions Sample 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