Metabolic,pathologic, and genetic analysis of prostate tissues: quantitative evaluation of histopathologic and mRNA integrity after HR‐MAS spectroscopy |
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| Authors: | Carissa F. Santos John Kurhanewicz Z. Laura Tabatabai Jeffry P. Simko Kayvan R. Keshari Akpene Gbegnon Romelyn DeLos Santos Scot Federman Katsuto Shinohara Peter R. Carroll Christopher M. Haqq Mark G. Swanson |
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| Affiliation: | 1. Department of Radiology, University of California, San Francisco, CA, USA;2. Department of Pathology, University of California, San Francisco, CA, USA;3. San Francisco Veterans Administration Medical Center, San Francisco, CA, USA;4. Department of Medicine, University of California, San Francisco, CA, USA;5. Department of Urology, University of California , San Francisco, CA, USA;6. Cougar Biotechnology, Los Angeles, CA, USA |
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| Abstract: | The impact of high‐resolution magic angle spinning (HR‐MAS) spectroscopy on the histopathologic and mRNA integrity of human prostate tissues was evaluated. Forty prostate tissues were harvested at transrectal ultrasound (TRUS) guided biopsy (n = 20) or radical prostatectomy surgery (n = 20), snap‐frozen on dry ice, and stored at ?80°C until use. Twenty‐one samples (n = 11 biopsy, n = 10 surgical) underwent HR‐MAS spectroscopy prior to histopathologic and cDNA microarray analysis, while 19 control samples (n = 9 biopsy, n = 10 surgical) underwent only histopathologic and microarray analysis. Frozen tissues were sectioned at 14‐µm intervals and placed on individual histopathology slides. Every 8th slide was stained with hematoxylin and eosin (H&E) and used to target areas of predominantly epithelial tissue on the remaining slides for mRNA integrity and cDNA microarray analysis. Histopathologic integrity was graded from 1 (best) to 5 (worst) by two ‘blinded’ pathologists. Histopathologic integrity scores were not significantly different for post‐surgical tissues (HR‐MAS vs controls); however, one pathologist's scores were significantly lower for biopsy tissues following HR‐MAS while the other pathologist's scores were not. mRNA integrity assays were performed using an Agilent 2100 Bioanalyzer and the electrophoretic traces were scored with an RNA integrity number (RIN) from 1 (degraded) to 10 (intact). RIN scores were not significantly different for surgical tissues, but were significantly lower for biopsy tissues following HR‐MAS spectroscopy. The isolated mRNA then underwent two rounds of amplification, conversion to cDNA, coupling to Cy3 and Cy5 dyes, microarray hybridization, imaging, and analysis. Significance analysis of microarrays (SAM) identified no significantly over‐ or under‐expressed genes, including 14 housekeeping genes, between HR‐MAS and control samples of surgical and biopsy tissues (5% false discovery rate). This study demonstrates that histopathologic and genetic microarray analysis can be successfully performed on prostate surgical and biopsy tissues following HR‐MAS analysis; however, biopsy tissues are more fragile than surgical tissues. Copyright © 2009 John Wiley & Sons, Ltd. |
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| Keywords: | cDNA microarray gene expression high resolution magic angle spinning (HR‐MAS) spectroscopy prostate cancer radical prostatectomy RNA integrity number (RIN) significance analysis of microarrays (SAM) transrectal ultrasound (TRUS) guided biopsy |
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