Journal of Histochemistry and Cytochemistry, Vol. 49, 1063-1064, August 2001, Copyright © 2001, The Histochemical Society, Inc.


BRIEF REPORT

New Molecular Approaches to Tissue Analysis

Ray B. Naglea
a Department of Pathology, University of Arizona Health Science Center, Tucson, Arizona

Correspondence to: Ray B. Nagle, Dept. of Pathology, University of Arizona Health Science Ctr., 1501 North Campbell Avenue, Tucson, AZ 85724-5724.


  Summary
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Summary
Introduction
Materials and Methods
Results
Literature Cited

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 49:1063–1064, 2001)

Key Words: genes, Human Genome Project, mRNA, protein sequences


  Introduction
Top
Summary
Introduction
Materials and Methods
Results
Literature Cited


  Materials and Methods
Top
Summary
Introduction
Materials and Methods
Results
Literature Cited

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 (Scott et al. 2000 ). Hematoxylin- and eosin-stained sections cut on a cryostat and mounted on charged glass slides were used for microdissection using the PixCell Laser Capture Microdissection Microscope (Arcturus Engineering; Mountain View, CA) essentially as described by Emmert-Buck et al. 1996 . Total RNA was extracted from laser captured cells using the Micro RNA Isolation Kit (Stratagene; San Diego, CA) (Luo et al. 1999 ).

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 (Luo et al. 1999 ). Ten µl of purified, resultant amplified RNA (aRNA) underwent second-round amplification using pdN6 random hexamers (1 µg/µl; Pharmacia, Piscataway, NJ) during first-strand synthesis and T7-oligo dT primer for the second-strand reaction, as previously described (Luo et al. 1999 ).

Second-round aRNA specimens were synthesized into randomly labeled {alpha}[32P]-dCTP probes using pdN6 random hexamers (1 µg/µl; Pharmacia) and the Superscript PreAmplification System reagents (Life Technologies) as previously described (Poirier et al. 1997 ). About 0.5 µg of target cDNA was crosslinked to the blot membrane with a UV Stratalinker 2400 (Stratagene). The positive control for each blot consisted of 0.5 µg of GAPDH. The negative control was a single-strand antisense laminin 5 ß3 cDNA. Reverse Northern blots were carried out as previously described (Poirier et al. 1997 ). In brief, 500 ng of target cDNAs including controls were denatured, combined with 111 µl sterile water, 80 µl 1 M NaOH, and 4 µl 0.5M EDTA, and crosslinked to an Ambion Bright Star Plus positively charged nylon membrane (Ambion; Austin, TX) using a Minifold II slot-blot apparatus (Schleicher & Schuell; Keene, NH). The membranes were prehybridized at 68C for 45 min in Perfect Hyb Plus solution (Sigma; St Louis, MO). Radiolabeled cDNA probe was denatured at 100C for 10 min, added to the prehybridization mixture, and hybridized overnight at 68C in Perfect Hyb Plus solution. The membrane was washed twice for 20 min each in 2 x SSC/0.1% SDS at 68C for 15 min. Higher-stringency washes were performed in sequence as necessary to remove background signals from the membrane, including two 0.2 x SSC/0.1% SDS washes at 68C for 15 min and two 0.1 x SSC/0.1% SDS washes at 68C for 15 min. The blots were then exposed on phosphor imaging screens (Molecular Dynamics; Sunnyvale, CA). Each blot was measured and plotted with a Phosphorimage 445SI scanner (Molecular Dynamics). Blots were stripped and re-probed up to four times.

Microarray Analysis
cDNA arrays were constructed on glass slides adapting the approach pioneered by Brown and colleagues (Schena et al. 1995 ; DeRisi et al. 1996 ; Brown and Botstein 1999 ). PCR-amplified cDNAs obtained from Research Genetics were spotted onto chemically activated glass slides in defined arrays using a OmniGrid DNA robotic Printer (Gene Machine, Genomic Instrument Services; San Carlos, CA) equipped with Telechem print heads and quills. Two type of arrays were utilized: (Scott et al. 2000 ) a 576-gene array comprised of 544 cancer-related genes, 24 housekeeping genes, and 8 ice plant genes used as controls (Emmert-Buck et al. 1996 ). A 5700-gene array was composed of 5184 genes and ESTs released as the first gene set by Research Genetic (gf200) plus an additional 516 genes collected from various research labs within the Arizona Cancer Center. Approximately 5 µg of mRNA isolated from cells or amplified from tissue microdissection was used for gene analysis. First-strand cDNA was made in the presence of Cy5-dCTP (red) or Cy3-dCPP (green). The two fluorescent first-strand cDNAs were mixed, denatured, and used as targets for the genes on the microarray slides. After hybridization and wash steps, emissions were collected using a Scan Array 3000 microarray reader (General Scanning, Packard Biochip Technologies; Billerica, MA) and quantitated using the Imgene 2.0 software (Biodiscovery; Los Angeles, CA).


  Results
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Summary
Introduction
Materials and Methods
Results
Literature Cited

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 (Hao et al. 2001 ). This information has provided an important indication that a post-transcriptional defect exists in prostate carcinoma. In the microarray experiments we were able to show that we could obtain enough RNA from as few as 200 cells for amplification and subsequent gene expression analysis using the microarray chips. Using the prostate carcinoma cell line (Du-145) we were able to analyze the effects on gene expression of laminin 5 (an important ECM molecule lost in prostate carcinoma) (Calaluce et al. 2001 ).


  Footnotes

Presented in part at the Joint Meeting of the Histochemical Society and the International Society for Analytical and Molecular Morphology, Santa Fe, NM, February 2–7, 2001.

Received for publication December 22, 2000; accepted February 16, 2001.
  Literature Cited
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Summary
Introduction
Materials and Methods
Results
Literature Cited

Brown PO, Botstein D (1999) Exploring the new world of the genome with DNA microarray. Nature Genet Suppl 21:33-37

Calaluce R, Kunkle MK, Watts GS, Schmelz M, Hao J, Barrera J, Gleason-Guzman M, Isett R, Fitchmum M, Bowden GT, Cress AE, Futscher BW, Nagle RB (2001) Laminin 5 mediated gene expression in human prostate carcinoma cells. Mol Carcinogen 30:119-129[Medline]

DeRisi J, Penland L, Brown PO, Bittner ML, Meltzer PS, Ray M, Chen Y, Su YA, Trent JM (1996) Use of a cDNA microarray to analyze gene expression patterns in human cancer. Nature Genet. 14:457-460[Medline]

Emmert–Buck MR, Bonner RF, Smith PD, Chaqui RF, Zhuang Z, Goldstein SR, Weiss RA, Liotta LA (1996) Laser capture microdissection. Science 274:998-1001[Abstract/Free Full Text]

Hao J, Jackson L, Calaluce R, McDaniel K, Dalkin BL, Nagle RB (2001) Investigation into the mechanism of the loss of laminin 5(a3B3g2) expression in prostate cancer. Am J Pathol 158:1129-1135[Abstract/Free Full Text]

Luo L, Salunga RC, Guo H, Bittner A, Joy KC, Galindo JE, Xiao H, Rodgers KE, Wan JS, Jackson MR, Erlander MG (1999) Gene expression profiles of laser-captured adjacent neuronal subtypes. Nature Med 5:117-122[Medline]

Poirier GM-C, Pyati J, Wan JS, Erlander MG (1997) Screening differentially expressed cDNA clones obtained by differential display using amplified RNA. Nucleic Acids Res 25:913-914[Abstract/Free Full Text]

Schena M, Shalon D, Davis RW, Brown PO (1995) Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 270:467-470[Abstract]

Scott KM, Fanta P, Calaluce R, Dalkin B, Weinstein RS, Nagle RB (2000) Diagnostic frozen prostate sextant biopsies: an approach for preserving protein and RNA for additional studies. Prostate 44:296-302[Medline]