BRIEF REPORT |
Correspondence to: Ray B. Nagle, Dept. of Pathology, University of Arizona Health Science Ctr., 1501 North Campbell Avenue, Tucson, AZ 85724-5724.
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Summary |
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The completion of the Human Genome Project will produce new opportunities for analysis of genes and their products in human tissue. The emergence of new technologies will enable investigators to directly examine human tissues for gene deletion, transposition, and amplification. In addition, we will be able to assess the complete gene expression of a tissue by examining the mRNA species using microarray chips. The emerging technologies of laser capture microdissection and RNA amplification enables these procedures to be carried out on groups of a few hundred cells, which will facilitate the examination of heterogeneous lesions. Finally, the application of tissue arrays and the capability of obtaining protein sequences in samples of only a few femtomoles of protein using desorption mass spectroscopy will revolutionize the analysis of protein expression.
(J Histochem Cytochem 49:10631064, 2001)
Key Words: genes, Human Genome Project, mRNA, protein sequences
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Introduction |
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Materials and Methods |
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Tissue Sampling
The objective of this study was to develop new methods of examining gene and protein expression in human samples of prostate tissue.To maximize the recovery of RNA and protein, transrectal biopsies and samples removed from radical prostatectomy were snap-frozen in liquid nitrogen cooled isopentane (
RNA Amplification and Reverse Northern Blotting Analysis
The purified RNA sample underwent amplification using 0.5 µg/µl T7-oligo dT primer, Superscript II Reverse Transcriptase (Life Technologies; Huntsville, AL), and Ampliscribe T7 Transcription Kit (Epicentre Technologies; Madison, WI) as previously described (
Second-round aRNA specimens were synthesized into randomly labeled [32P]-dCTP probes using pdN6 random hexamers (1 µg/µl; Pharmacia) and the Superscript PreAmplification System reagents (Life Technologies) as previously described (
Microarray Analysis
cDNA arrays were constructed on glass slides adapting the approach pioneered by Brown and colleagues (
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Results |
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Using snap-frozen clinical samples of prostate tissue, adequate RNA was obtained by microdissection for RNA amplification. Comparative hybridizations from two separate amplified samples verified that all mRNAs were equally amplified. Reverse Northern analysis using specific cDNA probes to integrin and ECM molecules confirmed that for several of these gene products the messages were present as previously suggested by ISH experiments, even though their respective proteins were not translated as evidenced by negative immunohistochemistry (
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Footnotes |
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Presented in part at the Joint Meeting of the Histochemical Society and the International Society for Analytical and Molecular Morphology, Santa Fe, NM, February 27, 2001.
Received for publication December 22, 2000; accepted February 16, 2001.
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Literature Cited |
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