Browsing by Author "Emmert-Buck, Michael R."

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    Assessment of normalization strategies for quantitative RT-PCR using microdissected tissue samples
    (2007) Erickson, Heidi S.; Albert, Paul S.; Gillespie, John W.; Wallis, Benjamin S.; Rodriguez-Canales, Jaime; Linehan, W. Marston; Gonzalez, Sergio; Velasco, Alfredo; Chuaqui, Rodrigo F.; Emmert-Buck, Michael R.
    Gene expression measurement techniques such as quantitative reverse transcriptase (qRT)-PCR require a normalization strategy to allow meaningful comparisons across biological samples. Typically, this is accomplished through the use of an endogenous housekeeping gene that is presumed to show stable expression levels in the samples under study. There is concern regarding how precisely specific genes can be measured in limited amounts of mRNA such as those from microdissected (MD) tissues. To address this issue, we evaluated three different approaches for qRT-PCR normalization of dissected samples; cell count during microdissection, total RNA measurement, and endogenous control genes. The data indicate that both cell count and total RNA are useful in calibrating input amounts at the outset of a study, but do not provide enough precision to serve as normalization standards. However, endogenous control genes can accurately determine the relative abundance of a target gene relative to the entire cellular transcriptome. Taken together, these results suggest that precise gene expression measurements can be made from MD samples if the appropriate normalization strategy is employed.
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    Global expression analysis of prostate cancer-associated stroma and epithelia
    (2007) Richardson, Annely M.; Woodson, Karen; Wang, Yonghong; Rodriguez-Canales, Jaime; Erickson, Heidi S.; Tangrea, Michael A.; Novakovic, Kristian; Gonzalez, Sergio; Velasco, Alfredo; Kawasaki, Ernest S.; Emmert-Buck, Michael R.; Chuaqui, Rodrigo F.; Player, Audrey
    Characterization of gene expression profiles in tumor cells and the tumor microenvironment is an important step in understanding neoplastic progression. To date, there are limited data available on expression changes that occur in the tumor-associated stroma as either a cause or consequence of cancer. In the present study, we employed a 54,000 target oligonucleotide microarray to compare expression profiles in the 4 major components of the microenvironment: tumor epithelium, tumor-associated stroma, normal epithelium, and normal stroma. Cells from 5 human, whole-mount prostatectomy specimens were microdissected and the extracted and amplified mRNA was hybridized to an Affymetrix Human Genome U133 Plus 2.0 GeneChip. Using the intersection of 2 analysis methods, we identified sets of differentially expressed genes among the 4 components. Forty-four genes were found to be consistently differentially expressed in the tumor-associated stroma; 35 were found in the tumor epithelium. Interestingly, the tumor-associated stroma showed a predominant up-regulation of transcripts compared with normal stroma, in sharp contrast to the overall down-regulation seen in the tumor epithelium relative to normal epithelium. These data provide insight into the molecular changes occurring in tumor-associated stromal cells and suggest new potential targets for future diagnostic, imaging, or therapeutic intervention.